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Molecular Systematics

Second Edition

Edited by
David M. Hillis

Craig Moritz

Barbara K. Mable

Sinauer Associates, Inc. Publishers

Sunderland, Massachusetts U.S.A.
Proteins: Isozyme Electrophoresis
Robert W. Murphy, Jack W. Sites, Jr., Donald G. Buth, and
Christopher H. Haufler

Protein electrophoresis, the migration of proteins under the influence of an elec-
tric field, is among the most cost-effective methods of investigating genetic phe-
nomena at the molecular level. Since the origin of starch gel electrophoresis
(Smithies, 1955) and the histochemical visualization of enzymes on gels (Hunter
and Markert, 1957),and the classic studies of H. Harris (1966),Hubby and Lewon-
tin (1966), and Lewontin and Hubby (1966), a major revolution in understanding
micro- and macroevolutionary processes has occurred. Using enzymatic and non-
enzymatic proteins, numerous investigations have focused on enzyme efficiency,
estimating and understanding genetic variability in natural populations, gene
flow, hybridization, recognition of species boundaries, and phylogenetic rela-
tionships, among other problems. The frequency of such investigations has not
waned in recent years, but rather has increased as refinements and new methods
have been developed.
Two general forms of protein data can be gathered simultaneously using elec-
trophoretic methods. One is derived from isozymes, which are all functionally
similar forms of enzymes, including all polymers of subunits produced by dif-
ferent gene loci or by different alleles at the same locus (Markert and Moller,
1959). The other data set consists of allozymes, a subset of isozymes, which are
variants of polypeptides representing different allelic alternatives of the same
gene locus. Both forms of data are important in molecular systematics, and both
involve proteins that can be separated on the basis of net charge and size.
The Cover
A circular tree of life inferred from ribosomal RNA genes, superimposed on the
genome of a triploid parthenogenetic species of gecko (Heteronotia binoei com-
plex). The chromosomes were stained by fluorescent in situ hybridization for the
ribosomal DNA arrays in a study of concerted evolution of repeated genes (see
Hillis et al., 1991, Science 251:308-310). The photographs represent a sampling of
multicellular species from the tips of the tree of life. Photographs by David Hillis.
Graphic design by Janet Young.

Moleculav Systematics, Second Edition

Copyright O 1996 by Sinauer Associates, Inc.

All rights reserved. This book may not be reproduced for
any purpose without permission from the publisher. For
information, address Sinauer Associates, 23 Plumtree
Road, Sunderland, MA 01375-0407 U.S.A.

FAX: 413-549-1118
Internet: publish@sinauer.com

Library of Congress Cataloging-in-Publication Data

Molecular systematics / edited by David M. Hillis, Craig Moritz,
Barbara K. Mable. -2nd ed.
P. cm.
Includes bibliographical references (p. ) and indexes.
ISBN 0-87893-282-8 (paper lay-flat bdg.)
1. Biology-Classification-Molecular aspects. I. Hillis, David
M., 1958- . 11. Moritz, Craig. 111. Mable, Barbara K., 1963-.
QH83.M665 1996
574.8'8--dc20 95-41159

Printed in Canada
5 4 3 2 1
52 Chapter 4 / Murphy, Sites, Bu th & Haufler

Here we provide a review of applications, they are attracted to neither the (positive) anode
step-by-step instructions on how to establish a nor the (negative)cathode.
horizontal starch gel electrophoresis laboratory, Uncharged amino acids are either non-polar
perform protein electrophoresis, stain for specific and hydrophobic or polar. These amino acids can
enzymatic and non-enzymatic proteins, and inter- become hydrogen-bonded to one another result-
pret the resultant gels. Although other methods of ing in folding (pstructure) or helical (a-helix) con-
protein electrophoresis exist, using media such as figurations, termed secondary structure. Depend-
cellulose acetate gels (Richardson et al., 1986),we ing on the primary and secondary structure, the
have chosen to detail horizontal starch gel meth- molecule usually undergoes additional folding to
ods because of their widespread use and effi- form its tertiary structure. The shape and size of a
ciency. Ways of avoiding or recovering from com- protein also may have an effect on protein migra-
mon pitfalls are described. The electrophoretic tion, depending on the pore size of the elec-
principles and methods described are applicable trophoresis matrix. To some extent the shape of a
to all organisms. particular protein is determined by the relative
Where possible, we provide inexpensive al- charges of adjacent amino acids because of the ef-
ternatives to costly equipment and methods, but fect of like charges repelling and different charges
not at the expense of increased health risk. As attracting. Finally, many proteins contain more
with most molecular methods used in systemat- than one polypeptide chain (subunit) bound to-
ics, some aspects of the data gathering pose ex- gether by hydrogen bonds, van der Waals forces,
treme health risks, both acute and chronic. There- ionic bonds, disulfide bridges, and/or hydropho-
fore, the appropriate level of caution, as known to bic interactions. Proteins having more than one
us, is always given the highest priority. polypeptide (multimeric) have a quaternary
structure (Darnel1et al., 1986).
Some forms of electrophoresis separate pro-
PRINCIPLES AND COMPARISON OF teins on the basis of net protein charge Q, shape
METHODS as measured by radius r, strength of the electric
field d, and viscosity of the suspension medium n,
as given by the following equation:
General Principles
Proteins are composed of amino acids joined by
covalent peptide bonds to form polypeptides.
These sequences, or "primary structures," are ge-
netically determined. Each of the 20 amino acids Under appropriate conditions, the rate of move-
has a unique side chain, characterized by its ment, u, increases with net charge and strength of
shape, size, and charge. The side chains on five of electric field and decreases with the size of the
these amino acids are either basic and thus posi- molecule. The actual situation is usually more
tively charged (NH3+;lysine, arginine and histi- complex than this simple equation indicates (e.g.,
dine), or acidic and negatively charged (COD; as- some proteins occur as relatively simple strands,
partic acid and glutamic acid). Charged side whereas others have a globular form) and indeed
chains are responsible for the movement of the much remains to be learned about the physics of
proteins through a matrix during electrophoresis. electrophoresis itself.
The net charge of each protein varies with pH; at All electrophoretic techniques consist of an
a low pH the amino groups become positively electric power supply, a support matrix (cellulose
charged, and at high pH the carboxyl groups be- acetate gel or strips, starch gel, etc.), and ionic
come negatively charged. Most proteins have a buffers. Electric current is applied to opposite
point at which the effect of positive and negative ends of the suspension medium via the ionic
charges are equal, the isoelectric point. Isoelectric buffers. Molecules (e.g., proteins) having a net
proteins do not move in an electric field because positive charge (cations) migrate to the cathode,
Proteins: Isozyme Electvophovesis 53

and negatively charged proteins (anions) migrate

to the anode. Following electrophoresis, the pro- Assumptions
teins may be visualized by a number of different The correct application of isozyme data requires
methods, the most frequently used being specific that banding patterns observed on gels are cor-
histochemical staining (Appendix 1 and refer- rectly interpreted. The most basic assumption that
ences cited therein). After electrophoresing a pro- evolutionary biologists make in using isozyme
tein sample in the gel matrix, the individual pro- data is that changes in the mobility of enzymes in
teins are selectively stained. Most of the stains an electric field reflect changes in the encoding
provide a specific substrate for the enzyme, allow DNA sequence. Thus, if the banding patterns of
it to catalyze the particular reaction involved, and two individuals differ, it is assumed that these dif-
then develop a dye that can be visualized in nor- ferences are genetically based and heritable (see
mal light or by fluorescence under UV light. Thus, Matson, 1984).Also, it is assumed that enzyme ex-
from the hundreds or thousands of enzymes in pression is codominant, i.e., that all alleles at a lo-
the crude extract, proteins with the same substrate cus are expressed. To interpret these banding pat-
utilization can be identified. terns, one must know something about the
As detailed later, numerous ionic buffers are number of subunits in the enzyme. Interpretation
available for electrophoresis. The buffers serve and non-heritable aspects of gel isozyme patterns
several functions. The primary function is to are detailed below and elsewhere (e.g., Richard-
buffer against pH change that occurs during son et al., 1986; Buth, 1990; Hernandez-Juviel et
electrophoresis: acid is produced at the anode al., 1992).In addition to biochemical components
and base at the cathode. The extent of pH change of gel interpretation, one must be aware of com-
is directly related to the duration of electro- partmentalization of enzymes, or enzyme activity,
phoresis, the voltage, and the current generated. in particular organs or organelles. For example,
Buffers also form an ionic connection between livers may have different enzymes than spleens or
the electric supply (electrodes) and suspension brains (see Murphy and Matson, 1986).In plants
medium (gel) and reduce the interaction of and animals, some enzymes are restricted to the
charged groups on the protein with any charged cytosol whereas others are found only in mito-
groups in the matrix, and may modify the net chondria or chloroplasts (Weeden and Wendel,
charges of proteins, carry enzyme stabilizers 1989; D.J. Crawford, 1990).
(e.g., disodium EDTA), or provide enzyme cata- For most enzymes, the genetic controls are
lysts (e.g., Mg2+). well enough known to allow genetic inferences to
The amino acid sequences of proteins are be made from gel isozyme patterns. The distribu-
changed by mutations in the encoding DNA lo- tion of isozymes per cell or tissue can be reliably
cus. Such mutations may alter shape and net predicted, homozygous and heterozygous indi-
charge, as well as catalytic efficiency and stabil- viduals can be identified, and conclusions about
ity (Shaw, 1965). Protein electrophoresis aims to genetic polymorphism, the breeding system of in-
reveal as many of these changes as possible. dividuals, and population structuring can be
However, considering the principles of elec- drawn. It is necessary to conduct breeding stud-
trophoresis and the properties of protein side ies only when banding patterns depart from ex-
chains, it is unlikely that all allelic variants will pectations. Cell fractionation studies can demon-
be identified using a single buffer pH, buffer sys- strate whether the enzymes are housed in the
tem, or concentration of gel, or even by using a cytoplasm or one of several separate organelles
single method of protein electrophoresis. Most (Weeden, 1983; Weeden and Wendel, 1989).
laboratories concentrate on manipulating the first Population geneticists have developed statis-
three of these four variables because of the diffi- tical models for interpreting genetic population
culty in mastering the alternative technologies structure (Chapter 10).The most relevant of these
and the added expense of additional equipment to gel interpretation is the Hardy-Weinberg equi-
and expendables. librium principle. Oversimplified,this states that
54 Chapter 4 /Murphy, Sites, Buth & Ha1

in the absence of selection, drift, and migration, Advantages of the four basic methods are com-
the frequencies of alleles in a randomly mating pared in Table 1, although choice of method will
population will maintain a stable equilibrium often be determined by availability of equipment
with genotype frequencies of AA = p2, Aa = 2pq, and expertise.
and aa = q2, where p is the frequency of allele A,
and q is the frequency of the alternative allele a. Starch Gel Electrophoresis (SGE)
Nonconformity to the prediction of Hardy-Wein- Hydrolyzed starch is heated in an ionic buffer so-
berg equilibrium indicates that the phenotypic lution and allowed to cool, thereby forming a gel.
variation has a non-genetic basis or that one or The ratio of starch to buffer can be varied to alter
more of the Hardy-Weinberg assumptions is not the size of the gel pores. Pore size allows for a
met in the population. Thus, for example, the indi- sieving effect in the gel. Thus, these gels can sepa-
viduals may not be randomly mating, or some nat- rate on the basis of both size (shape) and charge.
ural selective force may be acting on the species, or Two forms of SGE exist: horizontal and verti-
genes from neighboring populations may be mi- cal. In horizontal SGE, a poured gel is allowed to
grating into the study site. If these principles of bio- cool in a gel mold without further preparations.
chemistry, genetics, and gel interpretation are fol- Vertical starch gels are poured into double-sided
lowed, electrophoresis can yield many valuable molds having a "gel comb" or "well former" that
insights for the evolutionary biologist. makes the "gel wells" for holding tissue extracts
A major assumption in the use of allele fre- (Brewer, 1970; Morizot and Schmidt, 1990; see also
quency data to infer population structure is that Chapter 8). In general, the vertical system requires
alternative alleles at a given locus are selectively a greater amount of starch and larger quantities of
equivalent or neutral (Kimura, 1983a,b), or nearly tissue extract, allows for fewer samples to be run
neutral (Ohta, 1992). Exceptions to this assump- per gel, and is thus more costly. The advantages
tion are known (see below), and accepting neu- of vertical SGE include the avoidance of the phe-
trality for most protein polymorphisms also re- nomenon known as electrodecantation: as pro-
quires accepting largely untested or poorly tested teins migrate on horizontal gels, enzymes of high
null hypotheses. However, in the absence of evi- molecular weight tend to drop toward the bottom
dence for selection at a particular locus, it has of the gel. This may make slices from the upper
been suggested that studies begin with neutrality regions of the horizontal gel inferior or inade-
as a working assumption (Allendorf and Phelps, quate for resolving these proteins. Nevertheless,
1981). the method of horizontal SGE is used almost ex-
clusively in our laboratories and in the vast ma-
jority of other laboratories; the vertical system will
Comparison of the Primary Methods not be discussed further (for more information,
The four primary methods of electrophoresis dif- see Siciliano and Shaw, 1976; Morizot and
fer by the nature of the support medium: starch Schmidt, 1990).
gel (including both horizontal and vertical sys-
tems), polyacrylamide gel, agarose gel, and cellu- Polyacrylamide Gel Electrophoresis (PAGE)
lose acetate gel. Each method will be briefly de- Polyacrylamide gels are formed by the catalytic
scribed and discussed in terms of specific polymerization of monomeric forms of acry-
advantages and limitations. Less frequently used lamide and bisacrylamide. It allows the separa-
methods of resolving protein variants are not tion of proteins on the basis of both size and
discussed herein; these include paper elec- charge (Chrambachand Rodbard, 1971).The pore
trophoresis (Freifelder, 19821, isoelectric focusing size of acrylamide gels can be controlled by alter-
(Whitmore, 1990), immunoelectrophoresis, and ing concentrations of acrylarnide and/or bisacry-
two-dimensional electrophoresis (Harris and lamide. This sieving attribute has made PAGE one
Hopkinson, 1976; Hames and Rickwood, 1981). of the methods of choice in molecular biology lab-
Proteins: Isozyme Electrophoresis 55

Table 1
Comparison of the attributes of the four primary methods of protein electrophoresis
on gel support median

Separates by charge Yes Yes Yes Yes

Separates by size yesb yesb no no
Number of slices per gel >6b 1 1 1
Toxic nob Yes nob no
Running time 4-24 hr 4-6 hr 0.3-3 hrb 3-4 hr
Minimum amount of sample
required per gel 2 ~1 2 I41 0.5 plb 1I41
Maximum amount of sample
possible per gel >50 plb >50 plb 5 I41 >50 plb
Amount of stain required 5-50 ml 10-50 ml 1-3 mlb 10-50 ml
Electroendosmosis Yes nob Yes Yes
Voltage required (V/cm) 1-10 5-1 0 <3b 20
Cooling required Yes at times nob Yes
Gel easily handled usuallyb no yesb yesb
Simultaneously resolves cationic
and anionic proteins yesb no yesb yesb
Allows counterstaining of adjacent slices yesb no no no
'SGE = starch gel electrophoresis;PAGE = polyacrylarnide gel electrophoresis;CAGE = cellulose acetate gel electrophoresis;
AGE = agarose gel electrophoresis.
Perceived advantage.

oratories examining nucleic acid sequences be- number of variants identified (M.A. Riley et al.,
cause, unlike most other forms of gel elec- 1992).In addition, the large pores also cause elec-
trophoresis, it allows for the accurate, controlled troendosmosis, a "backwash of buffer solution
separation of charged particles on the basis of caused by gel charge groups that accelerates the
molecular weight (Chapters 8 and 9). General ref- mobility of cationic isozymes but retards or re-
erences to this system are found in Hames and verses the anionic isozymes. Although this prob-
Rickwood (1981). lem occurs with SGE and AGE, it is more pro-
nounced with CAGE (Harris and Hopkinson,
Cellulose Acetate Gel Electrophoresis (CAGE) 1976). CAGE has been discussed in detail by
Electrophoresis can be carried out on preformed Richardson et al. (1986).
cellulose acetate gels or strips. The gel form of cel-
lulose acetate is preferred because of repeatability Agarose Gel Electrophoresis
of experiments (Harris and Hopkinson, 1976). A (AGE) Agar and agarose gels are prepared much
major advantage is that electrophoresis can be car- in the same way as starch gels. Pure "agar" gels
ried out with very small quantities of tissue ho- have a relatively high concentration of acidic
mogenate. Although the gel itself is premade, it groups (carboxyls and sulfates), resulting in con-
must be soaked in the appropriate buffer prior to siderable electroendosmosis and occasional ad-
electrophoresis. Due to the large pore size, CAGE sorption of proteins, although adsorption prob-
has no sieving effect; proteins are separated on the lems may be overcome by use of highly purified
basis of net charge only and this may reduce the agarose (Harris and Hopkinson, 1976).
56 Chapter 4 / Murphy, Sites, Butlz & Haufler

low, 1988; Baker and Moeed, 1987; A.J. Baker,

APPLICATIONS AND LIMITATIONS 1992) have revealed that founder populations
generally have fewer alleles and lowered het-
Intraspecific Applications erozygosity than source populations. Easteal
(1985) conducted a similar study and estimated
Population Structure effective popultion size (N,) from both allozyme
Protein polymorphisms in natural populations frequency and ecological data for founder popu-
have been used to describe allele frequency lations of the giant toad (Bufo marinus) with
changes in both time and space. For example, known introduction histories in Hawaii and Aus-
Patarnello et al. (1989) showed GPI alleles to be tralia, and concluded that the former gave more
stable over a 15-year period in the marine amphi- accurate estimates.
pod Gammarus insensibilis (see below). A temporal Extremely low levels of allozyme heterozy-
study carried out by McClenaghan et al. (1985) on gosity in broad geographic surveys may imply
mosquitofish in the Savannah River drainage the occurrence of one or more recent severe bot-
basin showed that allele frequencies were gener- tlenecks, especially when genetically similar sis-
ally stable through time. ter species possess much higher levels of variabil-
On a spatial scale, population genetic struc- ity (e.g., Menken, 1987).However, for a number of
ture has been inferred from the geographic distri- theoretical reasons, heterozygosity (El) estimates
bution of allele frequencies. For example, among may not always be good indicators of past popu-
prairie dog populations (Cynomys ludovicianus), lation bottlenecks (Nei et al., 1975; Chakraborty
genetic variability was partitioned at several hier- and Nei, 1977; Motro and Thomson, 1982; see B.J.
archical levels that correlated with life history pa- Turner, 1984, for a possible empirical example;
rameters (Chesser, 1983). Rogers and Engstrom and Leberg, 1992, for a controlled study of bottle-
(1992) found a high degree of genetic structuring neck effects). From a conservation perspective, es-
in spiny pocket mice of the Liomys pictus species timates of allozyme variability may be used to
group, indicating that some species were para- make genetically based management recommen-
phyletic. In contrast, Shaklee (1984) surveyed dations for the recovery of endangered species
damselfish populations (Stegastes fasciolatus) col- (Vrijenhoek et al., 1985; A.F. Echelle et al., 1989;
lected from Midway Island to Hawaii, a linear Briscoe et al., 1992). Some plant studies (e.g.,
distance of about 2500 krn. Allele frequencies at all Muona et al., 1987) have demonstrated that the
eight polymorphic loci were remarkably uniform frequency of inbred individuals is significantly re-
(FST= 0.003; see Chapter 10) and, with the excep- duced as populations progress from seed to
tion of a few very rare alleles, all populations pos- seedling to adult stages.
sessed the same alleles at all loci.
Allozyme variability has been investigated Inbreeding, Outcrossing, and Dispersal
from the perspective of both space and time. For Allozyme studies can mesh genetic and ecological
example, Hoey and Parks (1991) examined allo- information to strengthen inferences about specific
zyrne variability and divergence of North Amer- aspects of population structure, especially breed-
ican and Asian plants of the genus Liquidambar ing structure and effective gene flow (e.g., Ham-
(Hamamelidaceae)and Haglund et al. (1993)eval- rick and Godt, 1984; Slatkin, 1985, 1987; Chepko-
uated Asian, North American and European nine- Sade and Halpin, 1987; Ryman and Utter, 1987;
spine sticklebacks (Gasterosteidae). Slatkin and Barton, 1989). Pope (1992) compared
Assessments of allozyme variability may be genetic estimates of gene flow and breeding struc-
used to infer historical events that have signifi- ture with expectations based on 10 years of census
cantly influenced the genetic structure of popula- data for two Venezuelan populations of the red
tions, such as bottlenecks (Bonnell and Selander, howler monkey (Alouatta seniculus). The two pop-
1974). Studies of several species of introduced ulations differed significantly in among-troop ge-
birds (Parkin and Cole, 1985; St. Louis and Bar- netic structure, with FST= 0.225 for population W
Proteins: Isozyme Electrophoresis 57

and FST= -0.142 for population G; the latter pop- 1983, 1985b, 1987; Knight and Waller, 1987;
ulation was subject to a higher rate of tmop failure Crouau-Roy, 1988; Van Treuren et al., 1991).
and recolonization. This pattern also correlates
with significant population differences in within- Paternity Studies
troop heterozygosity (FIs = -0.136 for W, FIs = Allozyme studies combining ecological data on
-0.064 for G), although troops in both populations dispersal with genotypic data that establish pa-
displayed excess heterozygotes, a pattern pre- ternity of offspring have allowed assessment of
dicted from ecological and behaviorial observa- the relative importance of several factors affecting
tions of mating and dispersion. The importance of genetic structure in some mammals. An experi-
obtaining independent estimates of dispersal can mental removal and colonization study of pocket
be illustrated by K.L. Brown's (1985) study of the gophers (Thomomys bottae; Patton and Feder, 1981)
demographic and genetic characteristicsof disper- revealed that migration (i.e., recolonization) was
sal in mosquitofish (Gambusia affinis) in a ther- nearly random with respect to the available
mally heated pond on the Savannah River Reser- source populations. This movement depressed be-
vation, South Carolina. Genotype frequencies of tween-field genetic heterogeneity, but this was re-
the dispersers were non-randomly distributed stored within a single generation due to a high
throughout the pond, and associations of genetic variance in male reproductive success. Juvenile
distance values and geographical distances be- dispersal apparently was responsible for the
tween collection sites indicated that the dispersers maintenance of intrapopulation variability in
did not constitute a random intermixing of refuge highly socially structured breeding groups of a
groups. Counter to intuitive expectations, disper- Neotropical cave-dwelling bat (McCracken and
sal in these populations resulted in an increase in Bradbury, 1977) and colonies of yellow-bellied
allelic differentiationbetween sites and an increase marmots (Schwartz and Armitage, 1980).Genetic
in mean levels of intrapopulation heterozygosity. markers revealed that inbreeding was avoided by
Very few plant or animal species have ade- the near-total dispersal of male offspring from
quate demographic data for estimating effective their natal colonies in both of these species.
genetic dispersal (Endler, 1979) and N,.In the Paternity of specific groups of offspring has
house sparrow (Passer domesticus), Fleischer (1983) been studied in several groups using detectable
used demographic data to predict FST(Wright, allozyme markers. For example, Tilley and Hans-
1943), and then tested the prediction with al- man (1976) collected female dusky salamanders
lozyme data and concluded that these birds ap- (Desmognathus ochrophaeus) and their broods, and
proximated a stepping stone model (Kirnura and showed that at least 7% of all individual clutches
Weiss, 1964) of genetic structure. However, a were sired by more than one male. Insemination
number of indirect approaches are now available and fertilization are therefore effectively uncou-
for inferring gene flow patterns entirely from the pled allowing the opportunity for sperm compe-
geographic distribution of allozyme frequencies, tition, which has been shown in controlled labo-
and these appear to be robust for some classes of ratory matings in D. ochrophaeus (Houck et al.,
dispersal patterns (reviewed by Slatkin, 1985, 1985).Other studies of colony breeding structure,
1987, 1993; see also Slatkin and Barton, 1989; based on either relatedness or parentage of spe-
Lessa, 1990; A.H. Porter, 1990; and Chapter 10). cific broods as inferred from allozyme markers,
Some population genetic surveys have re- include Evarts and Williams (1987), Harry and
vealed striking examples of heterozygote defi- Briscoe (1988),Quellar et al. (1988), and Price et al.
ciency, which could result from (1) strong selection (1989).Among studies of plants, Stanton (1986)
against heterozygous genotypes, (2) inbreeding, or summarized the ongoing problems in trying to as-
(3) a Wahlund (1928) effect (the inclusion of two or sess the contribution of different fathers to suc-
more genetically distinct units into a single popu- cessive generations, and Murawski and Hamrick
lation sample). In a number of studies, the in- (1990) investigated the effect of density of flower-
breeding explanation is favored (see OBrien et al., ing individuals on the mating systems of nine
58 Chapter 4 / Muvphy, Sites, Buth & Hauflev

species of trees. The latter authors discovered that plications (Daugherty et al., 1990; see also Crother,
there was (not unexpectedly) great variation 1992; T.E. Dowling et al., 1992).
among the density of flowering individuals,
which in itself varied annually, and in years of Ecological Genetics
lower flowering-tree density there was greater The neutral theory of molecular polymorphism
heterogeneity and/or more selfing. (Kimura, 1968; King and Jukes, 1969) has ques-
tioned the primacy of natural selection as an agent
Species Boundaries of molecular evolution (Lewontin, 1974; Nei and
Because isozyrne electrophoresis is a cost-effective Koehn, 1983). Several statistical studies have con-
method for screening a large number of single- cluded that most alternative alleles are selectively
copy nuclear gene loci (see Appendix I), it will equivalent and may represent transient stages of
continue to be especially helpful in multiple-pop- replacement, with fixation probability being a
ulation sampling efforts designed to determine function of mutation rate and effective population
species boundaries. Isozyme data readily can be size (see Kimura, 1983a,b).However, these studies
used as diagnostic markers in the sense described are based upon several largely untested assump-
by Davis and Nixon (1992; fixed differences be- tions, and may not distinguish among various
tween samples) for a priori identification of the ba- processes of neutrality and selection (W.B. Watt,
sic units (species) of phylogenetic analyses; this is 1985).Alternatively, several elegant multidiscipli-
especially critical in view of the potential for over- nary studies have investigated the selective basis
splitting taxa defined exclusively by rapidly for specific enzyme polymorphisms.
evolving portions of the animal mitochondria1 W.B. Watt (1985,1986) outlined a bioenergetic
genome (Moritz et al., 1992a).Isozyme studies of- approach for investigating possible functional and
ten reveal discordant geographic patterns be- ecological differences between alternate al-
tween levels of genetic divergence and taxonomic lozymes. Documentation of adaptive differences
boundaries inferred from morphological data, es- in allozymes requires demonstration of (1) differ-
pecially for geologically old and morphologically ences in a catalytic function, (2) allozyme-based
conservative radiations (see Wake et al., 1983; catalytic differences having physiological effects,
Wake and Larson, 1987). For example, Larson and (3) fitness differences in natural environments
(1989) has summarized 19 examples of pairs of between physiological effects (see also Koehn,
cryptic or morphologically very similar species of 1978; Powers, 1987). Watt (1977) identified four
plethodontid salamanders differing by from 1 to common GPI alleles in several natural butterfly
14 fixed allozyme markers, and similar cases are (Colias eu ytheme) populations, and demonstrated
known in other salamanders (e.g., D.A. Good et genotypic differences in survivorship, flight activ-
al., 1987; D.A. Good, 1989). Ranker and Schnabel ity, and mating success (Watt, 1983; Watt et al.,
(1986) used isozyme evidence to demonstrate the 1985,1986).Similar studies have been carried out
genetic differentiation of two lily species whose on bivalves (Koehn et al., 1980,1988; Koehn and
only clear separation was a difference in flower- Immermann, 1981; Koehn and Siebenaller, 1981;
ing time. J. Shaw et al. (1987) demonstrated the Hilbish et al., 1982; Hilbish and Koehn, 1985a,b;
clear genetic differentiation of two moss varieties, McDonald and Siebenaller, 1989), fishes (Powers
as did Odrzykoski and Szweykowski (1991) for et al., 1979; DiMichele and Powers, 1982a,b; Al-
the thallose liverwort. However, the reverse pat- lendorf et al., 1983; Leary et al., 1984; Crawford
tern is also well documented: morphologically and Powers, 1989; Ropson and Powers, 1989; Van
distinct taxa sometimes show little or no genetic Beneden and Powers, 1989; Ropson et al., 1990),
divergence (B.J. Turner, 1974; Echelle and Dowl- Drosophila (Heinstra et al., 1986; Barnes and Lau-
ing, 1992). Nevertheless, the many examples rie-Ahlberg, 1986; M.W. White et al., 1988), and
given above highlight the power of this approach marine amphipod crustaceans (J.H. McDonald,
for diagnosis of the basic units of analysis, and 1989; Patarnello and Battaglia, 1992). However,
these sometimes have profound conservation im- Eanes (1987) points out that many of these stud-
Proteins: Isozyrne Electrophoresis 59

ies were not designed to separate the effects of a Murphy et al., 1983a).At the other extreme, al-
single locus upon individual fitness relative to the lozyme divergence may have proceeded to the
contribution of linked loci; future efforts will re- point where too few electromorphs are shared,
quire strongly integrated approaches combining and many of those that are shared are convergent
in vivo and in vitro analyses and biochemical (e.g., Sites et al., 1984; Derr et al., 1987; Dimmick,
properties of segregating allozymes (Eanes et al., 1987).
1990). There are many groups, however, for which
allozyme divergence provides information appro-
Interspecific Applications priate for analysis of intra- or intergeneric rela-
tionships. Allozyme electrophoresis is appropri-
Phylogenetic Systematics ate for analyzing intergeneric phylogeny in birds
Allozyme data (and to a lesser extent, isozyme (Gutierrez et al., 1983; N.K. Johnson et al., 1988),
data) have been used extensively to investigate snakes (Murphy, 1988) and, occasionally, other
phylogenetic relationships. Some recent reviews groups as well (e.g., Hafner and Nadler, 1988).
of phylogenetic applications include M.W. Smith Most studies have focused on intrageneric rela-
et al. (1982, 1994) for vertebrates, Matson (1984) tionships (see reviews cited above), and these are
and N.K. Johnson et al. (1984) for birds, D.J. only informative when the individual loci are
Crawford (1989, 1990) for plants, and Kilias analysed as discrete characters (Murphy, 1993;
(1987) for lichens. Buth (1984a) reviewed the ap- Chapter 11).Among other things, this has the ad-
plication of isozyme and allozyme data to sys- vantage that the evidence for each node is made
tematic problems in general, and Mabee and explicit and therefore can be related back to the
Humphries (1993) and Murphy (1993) provide re- primary data (e.g., Crabtree, 1987; Sites et al.,
cent evaluations of methods and suggestions for 1990; Wiens and Titus, 1991).
phylogenetic analysis (see also Chapter 11). The
methods of data analysis used for phylogenetic Modes of Speciation
analysis of these data vary widely and are highly Allozymes can be used to explicate the patterns
controversial. Undoubtedly, the methods are still and mechanisms by which new species are
in a relatively early stage of refinement and much formed. Changes in allozyme frequency have
remains to be developed. What seems critical is been used to identify incipient species (Aradhya
that the assumptions associated with each et al., 1991; Gottlieb, 1973; McPheron et al., 1988),
method of analysis not be violated. Because each study sibling species (Anderson and Oakeshott,
method of analysis has its limitations, and some 1984), analyze how adaptation has contributed to
commonly used methods are simply invalid the process of speciation (Allegrucci et al., 1987),
(Buth, 1984a; Murphy, 1993; Chapter 111, the cita- differentiate between competing hypotheses for
tions in the general reviews (and below) should the origin of new species (Mayden, 1986; Small et
not necessarily be considered exemplary. Here, al., 19921, and explore the role that speciation
we restrict our comments to the use of allozymes; plays in evolution (Mindell et al., 1989, 1990).
isozyme characters such as the presence of dupli- Even though DNA analyses can be applied to
cate loci, patterns of gene expression, and the these kinds of questions, the ease with which elec-
ability to form heteropolymers are considered trophoresis can be used to survey large numbers
later, in the section "Gene Expression and Gene of individuals for genetically informative features
Duplication." makes allozyme analysis remain the tool of choice
Allozyme characters are subject to many of for many studies.
the same limitations as other forms of systematic
data. For example, morphologically distinct Paleobiogeography
species may show very low levels of divergence, Phylogenetic hypotheses formed from allozyme
and so differ by few phylogenetically informative data can be applied to answering questions of pa-
characters even when many loci are screened (e.g., leobiogeography. A primary method is Brooks
60 Chapter 4 / Murphy, Sites, Buth t3 Haufler

parsimony analysis, or BPA (Wiley, 1988a,b), also where evolutionary tempo can be examined from
known as co-speciation analysis. In the first step the perspective of cladograms (Mindell et al.,
of this method, cladograms are constructed for 1989,1990; Murphy and Lovejoy, 1995).
the taxa in question (Brooks, 1981,1990).Next, ge- Tempo questions may also be applied to (1)
ographic areas in which the species occur are des- estimated dates of dispersal or vicariance events,
ignated as if they were taxa. Using geological evi- (2) the relative arrival times of taxa on oceanic is-
dence, an area cladogram is constructed showing lands, (3) relative roles of colonization, extinction
the historical connections among the study areas. and historical factors in island biogeography, and
Next, the taxa are treated as if they comprised a (4) prediction of the time of origin of populations
completely polarized multistate transformation or geographic areas, such as islands, in the ab-
series in which each taxon and each internal sence of supporting geological data or radioiso-
branch of the tree are numbered. The taxa are tope dating, such as 14C.For example, Murphy
then coded using non-redundant linear coding (1983a)used genetic distance data from presumed
(O'Grady and Deets, 1987)and the species names sister taxa of a number of reptile taxa presumably
are replaced by their area names. A new area isolated by the same geological vicariant events in
cladogram is constructed based on the phyloge- Baja California, Mexico and found a good correla-
netic relationships of the species, and this new tion between geological dates and genetic similar-
area cladogram is presumed to represent the his- ity. Genetic distance data were then used to pre-
torical involvement of areas in the evolution of dict the age of one island in the Gulf of California,
the species. Although this is the preferred method Isla Santa Catalina. Unfortunately, most of the sis-
of paleobiogeographic analysis, it has rarely been ter taxa were presumed rather than tested using
applied to allozyme data (see also Kluge, 1988).A more preferable cladistic methods, such as that of
less preferable alternative is described below. Mindell et al. (1989, 1990). Nevertheless, genetic
similarity data were extended to test the applica-
Rates of Evolution bility of the MacArthur and Wilson (1963,1967)
Questions of evolutionary tempo are important theory of island biogeography as it relates to rep-
considerations, especially if applying the molecu- tiles on islands in the Gulf of California (Murphy,
lar clock (see Chapter 12) or examining relative 1983b),and to the colonization of islands by some
rates among different kinds of data from the same rattlesnakes (Murphy and Crabtree, 1985a).
taxa, for example, allozymes versus morphology.
Rosen and Buth (1980)provided a protocol for ex- Hybridization
amining evolutionary tempo using allozyme data Ideally, studies of interspecific hybridization
that included the calculation of ancestral genetic should incorporate three features, including: (1)
distance between all examined taxa and their hy- phylogenetic analysis of the taxa involved to al-
pothetical common ancestor. Murphy and Crab- low inferences to be drawn about the origin of the
tree (1985a) applied this method to rattlesnakes hybrid zone (primary versus secondary; e.g.,
and found that rates of divergence had been Hillis, 1985), (2) identification of autapomorphic
equal, although they were unable to confidently electromorphs in each of the hybridizing species,
calibrate the clock. However, comparisons of rela- which provides the most unambiguous genetic
tionships revealed by methods that assume equal markers for gene flow inferences (Murphy et al.,
rates (e.g., UPGMA) and distance (similarity) 1984), and (3) identification of at least three un-
methods that do not (e.g., distance-Wagner trees; linked markers (fixed or nearly fixed electro-
Chapter 11) most frequently reveal marked varia- morph differences) between hybridizing taxa (see
tion among lineages in rates of change (e.g., Chapter 2). With three or more single-copy mark-
Baverstock et al., 1979; Hillis, 1985).In general, the ers, F1 individuals (heterozygous for parental
evidence for an allozyme clock is weak (Avise and electromorphs at all markers) can clearly be dis-
Aquadro, 1982; Chapter 12). Finally, a phyloge- tinguished from most F2 or backcross classes,
netic approach has been developed recently which will be heterozygous for some but not all
Proteins: Isozyme Electrophoresis 61

markers (R.J. Baker et al., 1989; D.A. Good, 1989; in the hybrid origins of various unisexual taxa.
Wake et al., 1989; Arkvalo et al., 1993; Sites et al., Most carefully examined unisexual vertebrates
1993).Few of the studies conducted to date satisfy appear to be of hybrid origin (reviewed in Daw-
all of these criteria, but most have contibuted to a ley and Bogart, 1989).Typically unisexual taxa are
better understanding of the structure and dynam- characterized by higher levels of multilocus het-
ics of hybrid zones. Least informative are studies erozygosity than either parental form because of
in which hybridizing populations are not charac- their hybridity and the absence of segregation.
terized by any fixed allozyme differences (Green- Laboratory studies have confirmed patterns of
baum, 1981; Frykman and Bengtsson, 1984; clonal inheritance of fixed heterozygosity in some
Halkka et al., 1987). Hybridizing taxa distin- unisexual lizards (Dessauer and Cole, 1986). In
guished by several fixed differences (e.g., Patton cases of multiple ploidy levels among different
et al., 1984; D.A. Good, 1989; Szymura and Barton, unisexuals of hybrid lineages, allozymes fre-
1986,1991; Wake et al., 1989; Dessauer and Cole, quently show different staining intensities due to
1991) offer greater potential for inferring the ex- alleles that are represented unequally in the
tent and symmetry of introgressed nuclear genes. genome (Dessauer and Cole, 1984,1989; Dawley
Several recent studies have used allozyme et al., 1985; Kraus, 1991). Ideally, an analysis of
markers to infer the extent of introgression of suspected hybridization events should be carried
other classes of genetic markers (M.L. Arnold et out in a phylogenetic context that will permit the
al., 1987b; Harrison et al., 1987; Nelson et al., 1987; identification of uniquely derived (autapomor-
Marchant et al., 1988; R.J. Baker et al., 1989; Klier phic) markers in the parental species; this will
et al., 1991; Arkvalo et al., 1993), and occasionally eliminate ambiguities arising from the use of
hybridization is assessed in a historical context shared ancestral (symplesiomorphic) alleles to
(Dowling and DeMarais, 1993). Isozymes have define bisexual taxa involved in hybridization
also been used to study developmental stability as events (W.H. Wagner, 1983; Murphy et al., 1984;
manifested by morphological asymmetry (Gra- Funk, 1985; Moritz, 1987; Sites et al., 1990).
ham and Felley 1985), and the origin and distrib- Allozyme data are useful in the estimation of
ution of rare or unique alleles (called hybrizymes clonal diversity within gynogenetic or partheno-
by D.S. Woodruff, 1989; see also Hunt and Se- genetic populations which arise through recurrent
lander, 1973; Sage and Selander, 1979; Green- hybridization (Moritz et al., 1989c; Vrijenhoek,
baum, 1981; Barton et al., 1983; Case and 1989), mutation (Parker and Selander, 1976;
Williams, 1984; Murphy et al., 1984; Kocher and Spinella and Vrijenhoek, 1982), limited recombi-
Sage, 1986; Gollmann et al., 1988; Bradley et al. nation (Asher, 1970; Bogart et al., 1987), or some
1993),the genetic status of threatened and endan- combination of these factors. The matrilineal
gered taxa (Echelle and Conner, 1989; Dowling clones are frequently not a random representation
and Childs, 1992),and to address issues of hybrid of the possible genotypic diversity (B.J. Turner et
speciation (M.L. Arnold et al., 1990; Meagher and al., 1983);interclonal selection may produce habi-
Dowling, 1991; DeMarais et al., 1992). Future tat or trophic specialists, or hybridogens with dif-
studies of hybridization that merge ecological and ferent life history characteristics, and these kinds
molecular genetic approaches in appropriate phy- of differences may be "frozen" during the origin
logenetic and biogeographic contents offer great of new clones (Vrijenhoek, 1989). Others have ad-
potential for understanding processes involved in dressed questions of genome replacement in some
genome divergence, gene flow, and adaptation to of the unisexual salamanders (Spolsky et al., 1992)
alternative stable equilibria (Hewitt, 1988; Barton and have used isozymes in combination with
and Hewitt, 1989; Harrison, 1990). other genetic markers to provide evidence for
semi-independent segregation of unisexual al-
Parentage of Unisexual Biotypes lochthonous genomes during hybridigenetic
Allozyme electrophoresis is a powerful method meiosis in some populations of the salamander
for identifying the bisexual parent taxa involved genus Ambystoma (Kraus and Miyamoto, 1990).
62 Chapter 4 / Murphy, Sites, Buth & Haufler

Origin of Polyploid Plants (Ohno, 1970; MacIntyre, 1976; B.J. Turner et al.,
As in studies of hybridization, isozymes have 1980) can produce isozyme loci that often diverge
been valuable in identifying the parents of poly- markedly in their developmental expression
ploid plants. Isozymes have supported hypothe- (Whitt, 1981).Differences in gene number may ei-
ses based on other lines of evidence (Roose and ther serve as characters useful in systematic stud-
Gottlieb, 1976; Werth et al., 1985a) and differenti- ies (Gottlieb, 1982b; Whitt, 1983, 1987; Buth,
ated among alternative hypotheses (Holsinger 1984a), or have little value because of extensive
and Gottlieb, 1988; Gastony, 1986). Allozymes homoplasy (Sites and Murphy, 1991). These dif-
have shown that some polyploids have a single ferences can arise through gains of new genes
origin (Werth et al., 1985b) and that some are au- (duplication) or losses (gene silencing); both con-
topolyploids (Soltis and Rieseberg, 1986). In the ditions may be considered as derived relative to
process of exploring the origin of allopolyploids, an ancestral state. For example, many groups of
allozymes have been used to predict the existence fishes (Buth, 1983) and plants (Gottlieb and Wee-
of, and ultimately to discover, new diploid species den, 1979; Gottlieb, 1982a) have extra loci encod-
(Pryer and Haufler, 1993).Diversification and spe- ing enzyme systems, suggesting that gene dupli-
ciation in polyploid lineages have occurred cation events have played an important role in
through gene silencing (Werth and Windham, their evolution, perhaps in the acquisition of
1987; Gastony, 1991). However, if gene silencing novel gene functions (Ohno, 1970; Markert et al.,
regularly leads to diploidization of entire poly- 1975; Fisher et al., 1980). In fishes, tetraploidiza-
ploid genomes (Haufler, 1987), then the value of tion is followed by a shift from tetrasomic (pair-
allozymes for assessing ploidy must be ques- ing of homologous chromosomes in tetrads) back
tioned, especially in phylogenetically ancient to disomic (pairing in dyads) patterns of inheri-
groups. tance. During this "rediploidization," some 50%
of the duplicated loci are silenced either by fixa-
tion of new mutations or the deletion of some
Gene Expression and Gene Duplication codons (Allendorf and Utter, 1973; Ferris and
The expression of gene products is subject to both Whitt, 1977a,b; W.H. Li, 1980). Patterns of malate
temporal (ontogenetic) and spatial (cells/tis~ues) dehydrogenase (MDH) meiotic segregation dur-
variation in organisms. The predominance of ing rediploidization in the recently evolved
products of different L-lactatedehydrogenase loci tetraploid frog Hyla versicolor suggest polymor-
in different tissues of vertebrates (e.g., Ldh-A in phic (tetrasomic, disomic, and tetrasomic-dis-
skeletal muscle, Ldh-B in heart) is a classic exam- omic) inheritance thought to be a transitory phase
ple of this phenomenon (reviewed by Markert, between complete tetrasomy and complete dis-
1983). An example of the evolutionary conse- omy (Danzmann and Bogart, 1982a).A phyloge-
quences of regulatory divergence in gene expres- netic evaluation of the catostomid fish Moxostoma
sion is provided by the third L-lactatedehydroge- lachneri suggests that "retetraploidization" of the
nase locus (Ldh-C) in the bony fishes. Fishes of second glucose-6-phosphate isomerase locus, Gpi-
several morphologically primitive orders express B, is due to reactivation, or postpolyploidization
Ldh-C in many tissues, whereas in most teleosts regional duplication (Buth, 1982a).
Ldh-C expression is limited to eye or liver tissue Isozyme staining intensities may be used to
(Shaklee et al., 1973; Whitt et al., 1975; Shaklee investigate ploidy levels. Danzmann and Bogart
and Whitt, 1981).To ensure relevant comparisons (1982b) and Dessauer and Cole (1984) found that
of homologous gene products, extracts from ho- gene dosages, and thus ploidy levels (2n, 372, or
mologous tissues/organs must be prepared and 4n), could be inferred accurately from staining in-
specimens at similar developmental stages com- tensities because the subunit interactions were
pared. additive.
The duplication of genes via aneuploidy, As discussed earlier, many enzymes are mul-
polyploidization, and regional gene duplications timeric, composed of subunits that must be as-
Proteins: Isozyrne Electrophoresis 63

Table 2
Evolutionary patterns of creatine kinase gene expression in fishes
Character state

Character Ancestral Derived

Number of loci 2 (A, C) 4 (A, B, C, D)

Tissue specificity Widespread Restricted
Interlocus heterodimer formation Present Absent
Intralocus heterodimer formation Present Absent
Summarized from Ferris and Whitt (197813) and Fisher and Whitt (1978,1979).

sembled in order for the enzyme to function. Mul- and Murphy, 1991) and glucose-6-phosphate iso-
tiple isozymes of multimers can be produced by merase in the Leguminosae (Weeden et al., 1989)
combining different kinds of subunits in het- does not correlate strongly with phylogenetic re-
erozygotes (heteromers) and by the interactions lationships.
among multirners of duplicated genes in a multi-
locus isozyme system producing interlocus het- Limitations
eropolymers. Heteropolymer formation may be
non-random because regulatory differences may Taxonomic Limits
suppress the formation of some or all of the pos- Studies of population structure, breeding biology,
sible heteromers, e.g., the heterotetramers of L-lac- and other intraspecific applications require suffi-
tate dehydrogenase of some lizards (Gorman, cient levels of intraspecific variability. Allozymes
1971; Sites et al., 1986), fishes (Buth et al., 1980), are not sufficiently variable in some organisms,
and snakes (Murphy, 1988). making other molecular methods, such as RFLP
The isozyme characters (sensu Whitt, 1983, studies (Chapter 8), more appropriate. For exam-
1987; Buth, 198413; Murphy and Crabtree, 198513) ple, DeSalle et al. (1987b) examined the distribu-
of gene number, tissue specificity of expression tion of mtDNA haplotypes in populations of
(gene regulation) and posttranslational modifica- Drosophila mercatorum distributed along a short al-
tion, and heteropolymer assembly can be of sys- titudinal transect near Kamuela, Hawaii, and
tematic value only if they vary at a taxonomic found statistically significant spatial and tempo-
level useful to the investigator. These characters ral heterogeneity in the absence of isozyme diver-
may be useful for intraspecific, intrageneric, or in- gence. Intraspecific studies of birds are often ham-
trafamilial comparisons depending upon the pered by very low levels of isozyme polymor-
group (Buth, 198413).However, the few studies of phism (Barrowclough et al., 1985), yet Quinn and
enzyme systems reveal certain limited group White (198713) demonstrated extensive genomic
trends. Studies of creatine kinase (CK) expression DNA RFLP variability in the snow goose (Anser c.
in fishes by Ferris and Whitt (1978b), Fisher and caerulescens; see also Haig et al., 1993, for another
Whitt (1978,1979), and others permit the general- example). Similarly, Sites and Davis (1989) found
izations for CK isozyme characters listed in Table many more variable markers using restriction
2. Three of the four evolutionary patterns in Table sites in both mtDNA and nuclear ribosomal DNA
2 appear to hold for amphibians and reptiles than they found using allozymes among central
(Buth et al., 1985).In contrast, LDH expression in Mexican chromosome races of the lizard Sceloporus
sea snakes and cobras (Murphy, 1988), and the grammicus. These and other studies (Wetton et al.,
number of loci encoding glycerol-3-phosphate de- 1987; Karl et al., 1992; Karl and Avise, 1993) show a
hydrogenase (G3PDH) in squamate reptiles (Sites definite lower taxonomic limit to the resolving
64 Chapter 4 / Murphy, Sites, Buth & Haufler

power of protein electrophoresis (which may vary LIMITS TO DETECTION OF SEGREGATING ALLELES
among groups; Kessler and Avise, 1985b). Hubby and Lewontin (1966) recognized that gel
At the opposite extreme, some taxa have di- bands represented enzyme phenotypes, and not
verged to the extent that they share virtually no necessarily all underlying allelic variation. King'
alleles. For example, Sites et al. (1984) surveyed and Ohta (1975) introduced the term electro-
17 genera of batagurine turtles and found the morph to label allozymes of the same mobility as
taxa to be so divergent and homoplasy so exten- different classes of alleles. Allendorf (1977)
sive that they could not recover well-corrobo- stressed that electromorph identity did not mean
rated branches for most basal stems of the clado- identity in DNA base sequence; homology is a
gram. High levels of divergence found among conditional concept for isozyme phenotypes.
congeneric fern species average D = 1.1 (I = 0.33) Because accurate estimation of allelic varia-
(Soltis and Soltis, 1989), which approach or ex- tion has important implications for many evolu-
ceed the limits of resolution of isozyme elec- tionary questions (Coyne, 1982), the problem of
trophoresis. Nei (1987: 251-252) offered as a gen- hidden heterogeneity (G.B. Johnson, 1977) fos-
eral rule that if genetic distance D (Nei, 1972, tered several studies to determine how accurately
1978) is greater than 1.0, then the frequencies of conventional electrophoretic techniques estimate
back/parallel mutations will be high, and the genetic variability. R.S. Singh et al. (1976) used a
variance of D large, even if numerous loci are as- sequential assay of four different electrophoretic
sayed. The hierarchical taxonomic level at which conditions, termed sequential electrophoresis,
phylogenetic utility is lost (D 2 1.0) will vary and heat stability tests to examine Xdh-A variation
with taxonomic assignments and taxon-specific in Drosophila pseudoobscura. They resolved 37 al-
rates of molecular evolution-birds appear to be leles where only 6 had previously been identified
decelerated (Avise and Aquadro, 1982)-but gen- by conventional protocols. Other approaches to
erally the greatest phylogenetic utility for detecting cryptic alleles include thermostability
isozymes will be at the level of species or closely analysis (e.g., Chambers et al., 1981), peptide
related genera (Nei, 1987). mapping (Ayala, 1982), and the use of polyacry-
lamide gels of varying pore sizes to produce a
Sampling Limitations sieving effect for separation by size or molecular
Several kinds of limitations of isozyme tech- weight (G.B. Johnson, 1976,1979).
niques are recognized, including limits to the Although these methods show that conven-
number of (1) loci resolved, (2) alleles per locus, tional isozyme electrophoresismay underestimate
and (3) individuals required for population or variability, they do not reveal what proportion of
phylogenetic studies. The total number of loci alleles may remain undetected. Ramshaw et al.
that can now be visualized with histochemical (1979) examined several human hemoglobin vari-
staining techniques is in excess of 300 (D.A. ants of known amino acid sequence using both
Wright et al., 1983; Morizot and Siciliano, 1984; standard and sequential acrylamide electrophore-
Manchenko, 1994), but this is still only a very sis (varying conditions of pH and pore size).
small sample of the total genome. However, Three experiments determined what types and
given the size of most eukaryotic genomes (sum- proportions of substitutions could be resolved by
marized in Cavalier-Smith, 1985b; Loomis, 1988), these methods. First, 8 and 17 hemoglobin vari-
this is a constraint common to most molecular ants out of 20 were detected by the two proce-
techniques and will not be elaborated further dures, respectively. Second, groups of variants
here. What is apparent is that, in general, one with the same amino acid substitutions in differ-
needs to resolve about three times as many loci ent parts of the molecule were screened by two
as there are taxa in order to have a reasonable approaches and revealed 77% and 90% of the
chance of resolving most nodes of a cladogram in known variants, respectively. Third, 4 of 5 pairs of
a character-state evaluation. hemoglobins differing by charge-equivalent sub-
Proteins: Isozyme Electvophovesis 65

stitutions in the same positions were separated by Clearly, hidden heterogeneity is pervasive,
both procedures. There was no class of commonly and one cannot always rely on any single method
indistinguishable substitutions, and Ramshaw et to resolve all alleles. Equally important, however,
al. (1979) concluded that the standard protocol of are findings that (1) some loci are much more
electrophoresis was a powerful method for iden- likely than others to harbor cryptic alleles, espe-
tifying most variants. cially systems originally resolved as highly poly-
McLellan (1984) examined 14 whale myoglo- morphic by conventional methods, and (2) con-
bins of known sequence by sequential polyacry- ventional methods will resolve most or all
lamide electrophoresis (five pH values) and was variation at the more conservative loci. Further, a
able to separate 13 of the 14 variants. No further number of classes of studies will be largely unaf-
resolution was obtained by altering concentration fected by this phenomenon (Coyne, 1982). Fixed
or composition of the gels, or by screening with differences between populations or species de-
other techniques such as urea denaturation or iso- tected by conventional methods are real and the
electric focusing (McLellan and Inouye, 1986). differences can only increase by resolution of ad-
Aquadro and Avise (1982a) used several ditional alleles. Similarly, between-population al-
starch and acrylamide conditions, gel-sieving, iso- lele frequency heterogeneity is also real, regard-
electric focusing, and thermal stability tests to less of any underlying heterogeneity in
screen for cryptic alleles at three loci (sAat-A, electromorphs, because such differences in elec-
sMdh-A, and Est-1) in five populations of Per- tromorph classes should also reflect the same de-
omyscus maniculatus. sAat-A (their Got-1) was pre- viations of cryptic alleles. Other kinds of studies
viously known to segregate for two alleles across (e.g., absolute estimates of heterozygosity) may be
most of the range, sMdh-A (their Mdh-1) was es- more affected by cryptic allelic variability, but to
sentially monomorphic throughout the range, and an unknown degree. Obviously, any problem ad-
Est-1 was highly polymorphic, with eight alleles dressed with isozyme techniques will be better
resolved in earlier studies. None of the techniques understood by more accurate descriptions of al-
uncovered any further variation in either sAat-A lelic variation. Where time and resources permit,
or sMdh-A. In contrast, sequential electrophoresis we suggest that at least loci showing extensive
(five additional starch gel conditions) resolved 23 variation under standard conditions be screened
variants in Est-1, which were further resolved into sequentially with additional buffers to maximize
35 variants by heat denaturation, although the al- separation. Notwithstanding, at least one study
lelic nature of the latter group was not deter- (C.D. Chase et al., 1991) suggests allozymes corre-
mined. Aquadro and Avise (1982b) also uncov- late well with DNA RFLP data (Chapter 8). For
ered additional sMDH isozymes among ten phylogenetic studies involving extensive radia-
orders of birds using multiple buffers. tions likely including multiple monophyletic
Bradley et al. (1993) sequenced the entire cod- groups, conservative loci also should be screened
ing regions from multiple individuals of gophers with multiple buffers for resolution of additional
(Geornys) that expressed several combinations of electromorphs likely to define basal splits (S.B.
three Adh-1 electromorphs. They found that the Hedges, 1989; Burnell and Hedges, 1990). To this
three electromorphs were encoded by a total of six we would add that all hypothesized synapomor-
alleles at the nucleotide level and five alleles at the phic allozymes, identified as such through a pre-
amino acid level. However, each electromorph class liminary phylogenetic analysis, should be sub-
contained only alleles that were phylogenetically jected to sequential electrophoresis. However, the
closely related. Thus, the electromorphs represented sequencing study by Bradley et al. (1993; dis-
natural groups of alleles that would be expected to cussed above) lent support to the hypothesis that
be informative about phylogenetic relationships, even if electromorphs are encoded by different al-
even though all of the nucleotide variation was not leles at the nucleotide level, allozymes neverthe-
apparent in the allozyrnic differences. less are likely to represent related groups of
66 Chapter 4 / Murphy, Sites, Buth & Haufler

alleles, which are thus informative about evolu- complicate isozyme interpretations. Several non-
tionary relationships. Mendelian factors also may complicate isozyme
phenotypes via in vivo or in vitro environmental
NULL ALLELES AND ISOLOCI Other phenomena conditions, or through the action of modifier loci.
cause deviation from codominant expression of
allozymes. Null alleles (those with reduced or POSTTRANSLATIONAL MODIFICATIONS OF ENZYMES
no expression of a protein product) are detected Polypeptide synthesis involves (1) translation, (2)
by reduced staining intensity of some single polymerization, (3) termination, and (4) process-
isozymes on the same gel; complete absence of ing of the final protein product. Only the first step
activity may indicate null homozygotes (see involves the direct coding of nucleotide sequences
Utter et al., 1987).These interpretations are often into primary protein structure, while the others
ambiguous and require confirmation by breed- are posttranslational processes that give a final
ing studies (e.g., Stoneking et al., 1981). In the structure to the product. These latter processes
absence of breeding studies, quantification of a change the 20 primary amino acids specified by
null allele cannot be made reliably. Apparent het- the genetic code as monomeric building blocks in
erozygote deficiencies may be due to null het- polypeptide assembly into about 140 amino acids
erozygotes being scored as active homozygotes and derivatives in completed proteins (Uy and
(Foltz, 1986). Heterozygotes for null alleles are Wold, 1977). On gels, a number of these epige-
more readily detected if they either form partial netic events may produce conformational iso-
heteropolymer isozyrnes in polymorphic single- zymes, or multiple forms of a single gene product
locus systems (Burkhart et al., 1984) or are that differ in secondary or tertiary structure (also
expressed in multilocus, multimeric proteins called secondary isozymes or subbands; Rich-
(e.g., Engel et al., 1973; Allendorf et al., 1984; ardson et al., 1986) and/or variants that differ in
Utter et al., 1987; Gastony, 1991). In both cases, thermal stability (Lebherz, 1983). In some cases,
reduced intensities of one or more multiple modifying genes have been shown to be poly-
bands provide additional visual clues to the morphic for alleles that differ in their influence on
presence of null alleles. electrophoretic mobilities of the protein products
Another difficulty may occur when isozymes (Cochrane and Richmond, 1979; Womack, 1983;
with identical electrophoretic mobilities represent Dykhuizen et al., 1985).In other cases, altered mo-
the products of two different loci of the same mul- bilities appear to be restricted to specific tissues
tilocus enzyme system (Utter et al., 1987). These (Murphy and Crabtree, 1985b), or to be a function
isoloci may present rather complicated isozyme of environmental conditions and/or the physio-
patterns, and, if allelic variation is present, deter- logical state of the organism (McGovern and
mination of which locus is polymorphic (or Tracy, 1981; van Tets and Cowan, 1966; Fields et
whether both are) may not be possible. Isoloci al., 1989). For example, in a cryptic species of the
may be individually identifiable if their respective freshwater clam genus Corbicula, the synthesis of
encoded loci are synthesized at different levels in an enzyme seems to be a seasonal event in an en-
different tissues, but this appears to be uncom- tire population (Hillis and Patton, 1982). Such al-
mon (Allendorf and Thorgaard, 1984). Under terations of mobility may lead to incorrect hy-
some circumstances, different staining intensities potheses about the number of loci encoding an
are expected (seeUtter et al., 1987),but often such enzyme system (Hickey et al., 1989).
distinctions are difficult or impossible to make. Mobilities of some proteins are also suscepti-
Changing electrophoresis buffers often results in ble to protease degradation associated with re-
the separation of isoloci. peated freezing and thawing (Harris and Hopkin-
son, 1976; Richardson et al., 1986), or long- and
Other Sources of Phenotypic Variation of Isozymes short-term aging of the sample (Walter et al., 1965;
The phenomena described above may either limit Kobayashi et al., 1984).Moore and Yates (1983)
the resolving ability of isozyme electrophoresis,or showed that many of the loci frequently screened
Proteins: Isozyme Electrophoresis 67

in population and systematic studies were resis- respectively, as a function of varying enzyme
tant to mobility modification when kept at room dilution. This dilution effect, which occurs
temperature up to 12 hours after death. Posttrans- because GTDH molecules associate with one
lational effects can frequently be determined by another in the presence of coenzymes and purine
evaluating relative intensity of isozyme staining; nucleotides, is known from GTDH only. Al-
alternate segregating alleles usually give constant though the phenomenon of greater mobility with
patterns of expression, while breakdown effects increasing dilution occurs neither in all taxa nor
are likely to give a full range of expression of rela- on all buffer systems, it remains a variable to be
tive strengths (Richardson et al., 1986). considered.


between alternative nucleotide sequences within LABORATORY SETUP
a single gene locus has been investigated theoret-
ically by a number of workers with respect to its In order to carry out starch gel electrophoresis, a
potential evolutionary importance (W.B. Watt, number of pieces of equipment are essential and
1972; Strobeck and Morgan, 1978; Morgan and others are highly desirable. In many cases, initially
Strobeck, 1979; Golding and Strobeck, 1983). The more expensive alternatives are more cost-efficient
mechanism can generate new alleles at rates sev- in the long run because of time saved. Table 3 lists
eral orders of magnitude above standard gametic both the necessary and desirable equipment for
mutation rates if some minimum level of vari- SGE but does not include a detailed list of many
ability is already present at a given locus. This supplies (see Werth, 1985). Table 4 provides a list
hypothesis-namely, that "polymorphism gener- of chemicals required for the stain and buffer
ates polymorphism"-was empirically demon- recipes (Appendices 1 and 2, respectively).
strated by Ohno et al. (1969) in test crosses of 26 The room in which electrophoresis is to be
pairs of Japanese quail (Coturnix c. japonica) het- carried out should be equipped with sufficient
erozygous for different combinations of four alle- counter space and electric outlets, a large sink, gas
les for phosphogluconate dehydrogenase. They line (propane or natural gas), and a certified,
recovered 11 mutation-like events in enzyme working fume hood. If a fume hood is not avail-
phenotypes scored from 1011 test cross progeny, able, then all procedures involving the use of 2-
including new electromorphs, novel-combination mercaptoethanol and some alcohols must be
genotypes, and one case of the inheritance of avoided; the former liquid is highly volatile and
three alleles. If these results are d u e to the fumes may be lethal. Either the water pressure
intracistronic recombination, and if this is a gen- must be sufficiently high to allow for a faucet as-
eral phenomenon, then it may form part of the pirator or a vacuum pump or vacuum line must
explanation for the well-known "rare allele" be available. There should be an abundant supply
observations in many hybrid zones (Woodruff of distilled water and a water filter/deionizer.
and Thompson, 1980; Murphy et al., 1984; but see The starch gels must be cooled during elec-
Bradley et al., 1993). This type of recombination trophoresis. Ideally this is accomplished by per-
differs qualitatively from that between separate forming electrophoresis in a walk-in refrigerator,
loci by producing new gene products, which may chromatography chamber (dairy case), or stan-
then be transmitted in a Mendelian fashion, dard refrigerator; horizontal gels are cooled fur-
rather than new genotypic combinations that will ther using ice-filled aluminum trays. If a refriger-
be disrupted in later generations. ated chamber is not available, the ice levels must
be checked at least every 2 hours, making over-
NON-GENETIC VARIATION Frieden (1963) and night electrophoresis runs difficult. In addition,
Hernandez-Juviel et al. (1993) have reported rela- tissue homogenates are kept cool on ice while gel
tive mobility changes for glutamate dehydroge- loading takes place. Thus, an ice machine is
nase from bovine and rattlesnake liver extracts, highly desirable. If crushed ice is not readily avail-
68 Chapter 4 / Murphy, Sites, Buth & Haufler
Table 3
Basic equipment and non-chemical supplies necessary and desirable for starch gel
Quantity Quantity Protocol
Equipment Description needed desirable number

Major equipment
Freezer Manual defrost 1 1 6
Refrigerator >I2 cu ft 1 1 6 8
Analytical balance 0.1 mg to 100 g 1 1 1,2,6
pH meter 0.01 pH, with Tris probe 1 1 2,6
Fume hood 1 1 2,4,6
Water deionizer and filter 1 1 1,2,4,6
Power supplies 0-500 V; 0-100 rnA 1 lo+ 4
Refrigerated, high-speed >10,000 g 0 1 1
Centrifuge rotor Fixed angle; 24-36 place 0 1 1
Refrigerated chamber or 0 1 4
walk-in refrigerator
Ultracold freezer, -70°C 0 1 1
C02 (or LN2)backup for 0 1 1
ultracold freezer
Incubator 0 1 6
Tissue homogenizer High speed 0 1 1
Sonicator/cell disrupter 0 1 1
Water bath 0 1 6
Microwave oven 0 1 2,6
Pipetters, set Adjustable: 1 p1-5 ml O >I 1,3,6
Single lens reflex camera With macro lens and 0 1 8
yellow filter
Ice machine In lieu of blue ice
Minor equipment
Gel molds 1 >20
Buffer wells (trays) 1pair >20 pairs
Dessicators 2 4
Spatula (stainless steel) Large and small 12 of each >12
Magnetic stirrer Preferably with hot plate 1 1
Magnetic stirring bars Various sizes 1 pkg 1 pkg
Aspirator/vacuum line 1 1
Aspiration safety shield 1 1
Bunsen burner 1000 Cal 1 1
Heat gloves 1 pair 2 pairs
Gel slicer 1 1
Polystyrene stain boxes 10 >200
Hazardous chemical 1 2
disposal container(s)
Aluminum trays/blue ice
Timer 5-60 min

Proteins: Isozyme Electrophoresis 69

Table 3 (continued)
Basic equipment and non-chemical supplies necessary and desirable for starch gel

Quantity Quantity Protocol

Equipment Description needed desirable number

Forceps Small, straight tips 2 >4 3

Forceps Small, curved tips 2 >4 3
Dissecting kit Scalpel, scissors, etc. 1 2 1
Copy stand Adjustable height 1 1 8
Light box 1 1 6,s
Ultraviolet lamp Long wave (340 nm) 1 1 8
UV light face shield 1 1 8
Pipetter Ranging 1-10 ml; fixed
volume; bottle top 0 5 6
Liquid dispenser Adjustable; 10-60 ml 0 1+ 6
Erlenmeyer flasks 125-1 000 ml Numerous 2,6
Glass bottles, narrow 2004000 ml Numerous 6
mouth, amber color
Graduated cylinders 10-1000 ml 5 10 2,6
Beakers 3-3000 rnl 1 of each 12 6
Funnels Large and small 2 >2 2,6
Wash bottles 250 ml 1 6+ 1,5,6
Pasteur pipettes As needed 2,6
Disposable rubber or As needed 2,6
vinyl gloves
Disposable dust mask(s) As needed 2,6
'See text for discussion and alternatives.

able, then Blue IceTMpacks can be used during form, or a commercially available solvent (methyl-
electrophoresis without having deleterious effects. ene chloride containing dissolved plastic).
Most staining gels are placed in an incubator Table 4 lists the chemicals necessary to estab-
set at 37°C. Alternatively, gel staining can be car- lish an allozyme electrophoresis (specifically,
ried out in dark cabinets or drawers, the only ef- SGE) laboratory having a capacity to use many
fect being a longer stain reaction time. different buffer combinations for running and
It may be necessary or desirable to construct staining of most enzyme systems that have been
some of the equipment, especially gel molds, buffer adapted to eukaryotes.
wells, gel origin guide, gel slicer, slicing tray, and
aspiration shield. Plans and examples of equip-
ment are provided in Figures 1-5 and detailed as- PROJECT PLANNING
sembly instructions will be provided upon request
to R. W. Murphy. Buffer well plans are designed to The problems to be solved in preliminary studies
prevent accidental electrocution (see E.W. Spencer are (1)what is the optimal buffer system? and (2)
et al., 1966).Gel molds, buffer wells, and gel origin how does expression vary among tissues and
guides are constructed from high-quality acrylic which tissues are best for analysis? This technical
plastic (transparent polymethyl methacrylate) development phase can be combined with a pilot
sheets, such as Plexiglasm G. The pieces of plastic study (see Chapter 2) to determine the efficiency
are glued using either methylene chloride, chloro- of the approach. We have found that most fre-

1 -114 - 11
Figure 1 Plans for two types of gel molds used in
horizontal starch gel electrophoresis. (A) Simple gel
mold that requires the use of a sponge wick. (B) A
L wickless gel mold. The construction material is %-inch
-1 19 I+ acrylic plastic. All measurements are in millimeters.

quently the optimal gel buffer systems for partic- summarized commonly used combinations for
ular proteins vary among taxa and are not trans- plants.
ferable. Also, impurities in water can affect differ- With five gel setups, in a few days it is possi-
ences in electrophoretic conditions, making ble to determine optimal electmphoretic conditions
interlaboratory protocols vary. Unless multiple gel by surveying a few specimens for a wide array of
buffer systems are initially tried for each enzyme enzymes on virtually all commonly used gel buffer
or general protein system, much of the variation systems. Each of the five gels is made from a dif-
may be unresolved (see above). Before the ferent buffer and can be cut into 5 x 5 minislices, al-
isozyme data are gathered, it is highly desirable,
if not essential, to determine independently which
Figure 2 Design for an electrophoresis buffer tray,
of the various gel buffer systems are useful. that prevents accidental electrocution. (A) Base. (B)
Therefore, we have avoided suggesting buffer and Cover. Construction material is %-inch acrylic plastic.
stain combinations, although Kephart (1990) has All measurements are in millimeters.
(front Silicon


banana plug

u U

= 8 @ @ @

13 mm diameter holes
-T a e




0 @

3 5 . 5

3 ~ 5 3
72 Chapter 4 /Murphy, Sites, Buth & Haufler

6 mm steel rod Music wire

Groove to hold wire


1 Detail
of end

Figure 3 Gel slicing apparatus. (A) Bow slicer (con- in both number of loci and amount of allelic vari-
structed from %-inch aluminum bar). (B) Gel slicing
tray (constructed from %-inch acrylic plastic). AH mea- ability within loci (J.H. Gillespie and KoJima,
surements are in millimeters. 1968; Gottlieb, 1982a).
The final stage of planning involves the elec-
trophoresis of allozymes from numerous individ-
lowing the rapid survey of 30 or more enzyme sys- uals on established buffer systems and from
tems. Five minigels representing five different known tissues to generate data on allozyme vari-
buffers are simultaneously stained in the same ation. Gel runs must be well planned in advance
stain box making the protocol cost- and time-effi- in order to avoid unnecessa~remns.Richardson
cient. The specimens examined can represent the et al. (1986) have detailed many variables that
taxonomic diversity to be studied (see Chapter 2), a should be taken into consideration in the plan-
range of different tissue types, or both. ning stages. Some of the more important consid-
It may be important to have a mix of rela- erations are as follows:
tively rapidly and-slowlyevolving loci, especially
1. Enzyme systems sensitive to freezing and
if one study is to be compared to another, or if dif-
thawing (e.g., HBDH, G3PDH, IDDH, etc.)
ferent hierarchical taxonomic levels are being ex-
should be resolved first, preferably before
amined. Some enzymes, such as those involved
freezing the tissues or extracts.
with glycolysis, tend to be relatively conservative

Figure 4 Gel origin guide (constructed from %-inch

acrylic plastic). All measurements are in millimeters.
Proteins: Isozyme Electrophoresis 73

To vacuum

Detail of shield Setup

Figure 5 Gel aspiration setup. Plastic implosion shield
is made from %-inchor thicker acrylic sheet.
7. Drying agar overlays
2. Tracking dye (Appendix 2) or a blank space 8. Documentation of the results
should be used about every 10 specimens, but Many of the chemicals used in the various proto-
without separating populations or taxa. cols are extremely hazardous; Table 4 briefly
3. Initial specimen alignment on the gels should summarizes the known acute and chronic health
allow the first sample(s) to be repeated occa- hazards. For more information, "material safety
sionally, or at least on the end of the gel, espe- data sheets" may be available from suppliers free
cially if more than two taxa or populations are of charge upon request when ordering chemicals.
being surveyed. In order to differentiate Contact with these organic and inorganic sub-
among different alleles, side-by-side compar- stances should be avoided, as many are absorbed
isons are necessary. readily through the skin. Protocols should not be
performed without using protective laboratory
4. Some enzyme systems require, or are better coats, rubber gloves, dust masks, and eye goggles
resolved with, the addition of known activa- whenever appropriate. Food, drink, and tobacco
tors or cofactors such as EDTA and M ~ (e.g.,
~ + should not be allowed in the laboratory. Other
PGM), or coenzymes such as NAD (e.g., safety precautions, such as eye wash stations,
ADH), or NADP (e.g., GGPDH, PGDH) (Har- showers, chemical spill clean-up kits, and first
ris and Hopkinson, 1976). These are added to aid kits, should be available. Operator safety
specific gels either before cooking (EDTA, must be accorded priority over all other consid-
MgZ+)or following aspiration (coenzymes). erations.

PROTOCOLS Pro.tocol1: Ti5su.e Homogenization

(Time: 2 min/specimen)
1. Tissue homogenization
2. Preparation of starch gels Tissue extract preparation may precede or follow
preparation of the starch gels depending on (1)
3. Gel loading the necessity for extremely fresh extracts, or (2)
4. Electrophoresis desirability of preparing gels a day in advance.
The homogenization of tissue samples far in ad-
5. Gel slicing vance of their use may result in significantly re-
6. Histochemical staining duced levels of enzyme activity. Many extraction
74 Chapter 4 / Murphy, Sites, Buth & Haufler

Table 4
Chemicals required for electrophoresis, use, location of storage, and health hazard information
Chemical (use)a ~ o c a t i o n ~ numberC Health and safetyd

Acetic acid, glacial (buffers) s

Acetone (RDH) s
Cis-Aconitic acid (ACOH) f
Adenosine (ADA) r
Adenosine 5'-diphosphate (AK, ARK, CK, ENO, PK, TAT) f
Adenosine 5'-monophosphate (AK) f
Adenosine 5'-triphosphate (AK, GLAL, GUK, HK, PFK, f
Agar (general) s
D~-Alanine(ALAT, ALPDH) s
DL-Alanyl-DL-methionine (PEP) f
Aldolase (GAPDH, PFK) r
Amaranth (tracking dye) s
3-Amino-9-ethylcarbazole(PER) s
N-(3-Aminopropy1)-diethanolamine(buffers) s
N-(3-Aminopropy1)-morpholine(buffers) s
Ammonium hydroxide (general) s
Arsenic acid (Na salt) (ADA, GAPDH, TPI) s A-6756
Ascorbic acid (GLT) s A-1417
L-Ascorbicacid (GLAL, NTP) s A-0278
L-Aspartic acid (AAT) s A-9256
1,3-Bis(dimethylamin0)-2-propanol(buffers) s B-4298-5
Boric acid (buffers) s B-0252
Brilliant blue G (CBP, GP) s 8-1131
Calcium chloride (GUK, PER) s C-3881
Citric acid (anhydrous, free acid) (buffers) s C-0759
Citric acid (dihydrate) (buffers) s C-7254
Citric acid (monohydrate) (buffers) s C-7129
4,6-Diamidino-2-phenylindole(DAPI) f D-1388
o-Dianisidine dihydrochloride (PEP) r D-3252
Dichlorophenol-indophenol (CBR, DDH, GR, LGL) s D-1878
Dihydroxyacetone phosphate (TPI) r D-7878
Dowex (ion exchange resin) (TPI) s 50 x 4-200R
N,N-Dimethylformarnide (PER) s D-4254
Dimethyl sulfoxide (P GA, P GALA) s D-5879
Ethanol (ADH, ODH) s xxxxe
Ethylenediaminetetraacetate s EDS
(EDTA) free acid (buffers, AAT)
EDTA-dihydrate (PGAM, SUDH, buffers) s
Fast blue BB (salt) (ALAT, AAT, EST) f
Fast blue RR (salt) (ALP) f
Fast garnet GBC (salt) (PGLUR, CAP) f
Flavine adenine dinucleotide (GR) f

Proteins: Isozyme Electrophoresis 75

Table 4 (continued)
Chemicals required for electrophoresis, use, location of storage, and health hazard information
Chemical (use)a ~ o c a t i o n ~ numberC Health and safetyd

Fluorescein diacetate (CA) f

Formaldehyde (FDH) s
D -Fructose-1,6-diphosphate (ENO, FBA, FBP, ALD, f
D-Fructose-6-phosphate (GPI, PFK, buffers) f
Fumaric acid (FUMH) s
D - Gluconic acid lactone (HADH) s
D (+)Glucose (AK, ARK, CK, GCDH, HK, PK) s
a-D-Glucose-1,6-diphosphate (PGM, UGUT) f
a-D-Glucose-1-phosphate (PGM) f
D - Glucose-6-phosphate (G6PDH) f
Glucose-6-phosphate dehydrogenase (AK, CK, GPI, HK, r
L -Glutamic acid (GLAL, GTDH) s
L-Glutamic dehydrogenase (ALAT, TAT) r
Glutathione, oxidized (disodium salt) (GR) f
Glutathione, reduced (FDH, HAGH, LGL) r
Glyceraldehyde-3-phosphate dehydrogenase (ALD, FBA, r
DL - Glyceric acid (GLYDH) s
Glycerol (fixative) s
DL -a-Glycerophosphate (G3PDH) r
a-Glycerophosphate dehydrogenase (PGK) r
Glycine (buffer) s
Glycolic acid (HAOX) s
Glycyl-L-leucine(PEP) f
Glyoxalase I (HAGH) r
Guanine (GDA) s
Guanosine-5'-monophosphate (GUK) r
1-Hexanol (ADH) r
Hexokinase (AK, ARK, CK, PK) f
L-HistidineHC1 monohydrate (gel buffer) s
Hydrochloric acid s
Hydrogen peroxide (CAT, PER) r
DL - P-Hydroxybutyric acid (Na salt) (HBDH) r
Hypoxanthine (XDH) s
Inosine (PNP) s
Inosine triphosphate (NTP) f
DL-Isocitricacid (IDH) r
Isocitric dehydrogenase (ACOH) f
a-Ketoglutaric acid (ALAT, AAT, TAT) r
DL -Lactic acid (Buffer, LDH) s

76 Chapter 4 / Murphy, Sites, Buth 63 Haufler

Table 4 (continued)
Chemicals required for electrophoresis, use, location of storage, and health hazard information
Chemical (use)" ~ o c a t i o n ~ numbe? Health and safetyd

L-Lactic dehydrogenase (AK, ALAT, CK, ENO, GUK,

L -Leucyl-L-alanine(general PEP)
L -Leucylglycylglycine (PEP)
L -Leucine P -naphthylamide HC1 (CAP)
Lithium hydroxide (buffer) '
Magnesium acetate (CK)
Magnesium chloride (general)
Magnesium sulfate (ALP, PK, buffers)
Maleic acid (buffers)
D L-Malic acid (buffer, MDH, MDHP, ME)
Malic dehydrogenase (FLTMH)
D -Mannose-6-phosphate (MPJ)
2-Mercaptoethanol (FBP, NTP, PFK)
Methylglyoxal (HAGH, LGL)
Methyl alcohol
4-Methylumbelliferyl acetate (EST)
4-Methylumbelliferyl-N-acetyl-P-D- galactosamide (P GAI
4-Methylumbelliferyl-N-acetyl-P-D-glucosamide (P GA)
4-Methylumbelliferyl-a-D-galactoside (aGAL)
4-Methylumbelliferyl-P -D - galactoside (P GAL)
4-Methylumbelliferyl-P-D-glucoside(P GLUS)
4-Methylumbelliferyl-P-D-glucuronide (P GLUR)
Molybdic acid ammonium tetrahydrate (GLAL)
MTT (tetrazolium salt) (general)
P-NAD (Nicotinamide adenine dinucleotide) (general)
P-NADH (general)
P-NADP (general)
Naphthol AS-BI P -D-glucuronicacid (P GLUR)
Naphthol blue black (amido black) (GP)
a-Naphthyl acetate (EST)
P-Naphthyl acetate (EST)
P-Naphthyl acid phosphate (ALP)
Nitro blue tetrazolium (NBT) (general)
Nucleoside phosphorylase (ADA)
1-Octanol (ADH, ODH)
D -0ctopine (OPDH)
1-Pentanol (ADH)

Proteins: Isozyme Electvophovesis 77

Table 4 (continued)
Chemicals required for electrophoresis, use, location of storage, and health hazard information
Chemical (use)' ~ o c a t i o n ~ numberC Health and safewd

Peroxidase (PEP)
Phenazine methosulfate (PMS) (general)
Phenolphthalein diphosphate (ACP)
L -Phenylalanyl-L-proline(PEP)
Phosphocreatine (CK)
Phospho(eno1)pyruvate(AK, GUK, PK, LTK)
Phosphoglucomutase (UGUT)
6-Phosphogluconic acid (Ba salt) (PGDH)
6-Phosphogluconic acid (trisodium salt) (PGDH)
Phosphoglucose isomerase (FBP, MPI)
(= Glucose-6-phosphate isomerase)
3-Phosphoglyceric phosphokinase (PGAM)
Phosphoglycerate mutase (ENO)
D (+I-2-Phosphoglyceric acid (ENO, PGAM)
D (-)-3-Phosphoglyceric acid (ENO, PGK)
Polyvinylpyrrolidone (ALP, AAT)
Potassium acetate (CK)
Potassium bicarbonate (buffer)
Potassium chloride (AK, ENO, GUK, PK, buffers)
Potassium cyanide (RDS)
Potassium hydroxide (XDH)
Potassium iodide (CAT)
Potassium phosphate (dibasic-anhydrous) (buffer)
Potassium phosphate (dibasic- trihydrate) (buffer)
Potassium phosphate (monobasic) (buffer)
Potassium sulfate (UK)
Pyrazole (GLYDH, HADH, general)
Pyridoxal-5'-phosphate (TAT)
L -Pyroglutamic acid (PCDH)
Pyrophosphate (UGUT)
Pyruvate kinase (salt free) (AK, CK, ENO, GUK, UK)
Pyruvic acid (ALPDH, HAGH, GLYDH)
Retinol (RDH)
Shikimic acid (SKDH)
Sodium acetate (ACP)
Sodium chloride (ALP, HBDH)
Sodium hydroxide (buffer)
Sodium phosphate (Na2HP04)(buffers, AAT)
Sodium thiosulfate (buffers, CAT, TST)
D-Sorbitol (buffers, IDDH)
Starch (potato), hydrolyzed
Succinic acid (free acid) (buffer)

78 Chapter 4 /Murphy, Sites, Buth & Haufler

Table 4 (continued)
Chemicals required for electrophoresis, use, location of storage, and health hazard information

Chemical (use)' ~ o c a t i o n ~ numberC Health and safetyd

Succinic acid (disodium salt) (SUDH) s S-2378 E, S, I

Sucrose (buffers) s S-9378 E, S, R
5-Sulfosalicylicacid (CBP, GP) s S-2130 E, S, R, I
Sulfuric acid (GLT) s D, V
Trichloroacetic acid (CBP, GP)
Triethanolamine (buffers)
Triosphosphate isomerase (PFK)
Trizma base (buffers)
L -Tyrosine HC1 (TAT)
Uridine 5'-diphosphoglucose (UGUT)
Uridine 5'-monophosphate (UK)
Venom, Crotalus atrox (PEP)
Xanthine (XDH)
Xanthine oxidase (ADA, GDA, PNP)
Zinc chloride (HAGH)

a Enzyme system(s) and/or buffer($.

s = room temperature, shelf; f = freezer (-20°C); r = refrigerator (04°C).
Sigma Chemical Company (St. Louis, Missouri, U.S.A.) catalog numbers are provided to distinguish among multiple forms of
some chemicals; however, reagent-grade chemicals may be obtained from most major suppliers. Sigma catalog numbers are pro-
vided as a reference and do not necessarily represent endorsement.
Safety information as of 1993 from information available from Sigma. All of these chemicals may be harmful if inhaled, swal-
lowed, and/or absorbed through skin. A = allergen (especially respiratory tract and skin); B = effects on fertility (e.g., spermatoge-
nesis, testes, epididymis, sperm ducts, male fertility index and/or post-implantation mortality); C = carcinogen; D = high concen-
trations are extremely destructive to tissues (mucous membranes, respiratory tract, eyes and skin); E = eye irritant; F = potentially
fatal (if inhaled, swallowed, or absorbed through skin), at acute level; G = CNS depression, narcotic effect, convulsions, etc.; H =
headache, nausea and/or vomiting; 1 = not thoroughly investigated; M = mutagen; N = no hazards known; 0 = other harmful
effects known; P = poisonous; R = respiratory irritant; S = skin irritant; T = teratogen; V = corrosive.
Denatured alcohol should not be used. We have achieved greatest ADH enzyme activity by using "gold" tequila as a source of
ethanol; other liquors may work equally well.

buffer recipes use 2-mercaptoethanol, a sulfhydryl mogenizer, (3) hand grinding with a glass test
reducing agent, to reduce subbands. However, at tube or rod sanded on its base and a porcelain
least in reptiles, this ingredient significantly re- spot plate (Werth, 1985; Kephart, 1990), (4) motor-
duces the activity levels of many enzyme systems. ized plastic (e.g., TeflonTM)pestle and plastic (cen-
Phenolic compounds in many plant tissues trifuge tube) mortar, or (5) a high-speed tissue ho-
form complexes with proteins upon homogeniza- mogenizer with a generator blade (Figure 6).
tion. The addition of polyvinylpyrrolidone to the Homogenization using devices designed not to
extraction solution usually reduces this problem; disrupt cell membranes (Figure 6) may require
some plants also require other ingredients (see that the samples be subjected to sonication or re-
Werth, 1985; Kephart, 1990). freezing for 10 min at -20°C. All of the methods
There are several ways of extracting enzy- work very well, even without sonication; the lat-
matic proteins from cells including (1)simple ter, and initially most expensive method, is the
maceration of tissue(s) with scissors followed by fastest. If samples are not to be used immediately,
freezing, (2) use of a hand-held ground-glass ho- refreeze, preferably in an ultracold freezer.
Proteins: Isozyme Electrophoresis 79

preferably at >10,000 g for 15-30 rnin, to sepa-

rate extracted proteins from cellular debris.
Although highly desirable, centrifugation is
not always necessary for some tissues and
some taxa.

Protocol 2:Preparation of Starch Gels

(Time: 2-3 hr/gel)
Gel cooking involves either the boiling of hy-
drolyzed starch in gel buffer below) or the addi-
tion of starch to hot gel buffer (e.g., Micales et al.,
1986).Hydrolyzed potato starch may be made fol-
lowing the method of Smithies (1955) or pur-
chased. Although relatively expensive, Con-
naught Medical Laboratories (Toronto, Ontario,
Canada) starch has a longstanding reputation for
.consistently producing very high-quality gels.
Electrostarch Co. (Madison, Wisconsin) starch is
relatively inexpensive, but variable in quality, and
sometimes requires the addition of Connaught
starch to make it usable. Starch from Starch Art
Corp. (P.O. Box 268, Smithville, Texas 78957
U.S.A.) produces highly satisfactory gels and is
moderately priced. As with Electrostarch, a free
sample is available on request. Other sources of
Figure 6 Homogenization of tissue extracts using a hydrolyzed potato starch include varisus chemi-
high-speed homogenizer. See text for other methods. cal (e.g., Sigma) and biological supply companies;
these are invariably the most expensive and usu-
1. Dissect out desired tissues or retrieve previ- ally obtain their stock from the sources above.
ously dissected tissue samples from the freezer Typically, starch gels are made in concentra-
and place them in a clean grinding tube. tions of 9-18% (w/v) starch in gel buffer, depend-
ing on the quality of starch, preferred texture of
2. Dilute the samples 3-5 fold with grinding so- the gel, and desired sieving effect obtained d k g
lution. The ice-cold grinding solution may be electrophoresis. The appropriate concentrations
either distilled, deionized water or one of are determined by trial (and error).
many solutions described in the literature Three problems may occur during gel prepa-
(e.g., Selander et al., 1971; Harris and Hop- ration: undercooking, overcooking, and burning.
kinson, 1976; Werth, 1985; Kephart, 1990). If Undercooking can be recognized by soft, wet gels
enzyme activity levels are to be surveyed, the that are difficult to handle following slicing; un-
tissue samples must be weighed precisely and dercooking is rare. Overcooking is easily recog-
diluted (Klebe, 1975; Kettler and Whitt, 1986; nized during four stages: aspiration, cooling,
Kettler et al., 1986). loading, and slicing. During aspiration, over-
3. Mechanically homogenize the mixture of tis- cooked gels may boil out of the flask. Vigorous
sue and grinding solution. The mixture shaking during aspiration may be required if the
should be kept ice-cold during the homoge- gel is to be saved, although this is sometimes in-
nization process. effective. During cooling, deep crevasses or circu-
4. Just prior to use, centrifuge the homogenate, lar or octagonal patterns may form in the surface.
80 Chapter 4 / Murphy, Sites, Buth & Haufler

Overcooked gel mixtures tend to stick to the gel a. While wearing eye protection and insulated
molds, often splitting during loading or removal glove($, continuously swirl flask above a 1,000-
for slicing following electrophoresis; gel slices are Cal Bunsen burner (Figure 7A). The mixture
tacky and sometimes impossible to handle. Burn- will become viscous and then quite rapidly
ing can occur without overcooking. It results from much less viscous. As boiling begins (after
not swirling the mixture vigorously enough dur- about 3 4 min) stop heating.
ing cooking and can be recognized by brown- b. Use a magnetic stirring hot plate and large mag-
black, burned starch on the bottom of the flask netic stirring bar to heat the starch-buffer mix-
and/or dark flecks in the gel. Burning frequently ture until the mixture becomes too viscous for
results in tacky gels. Improperly cooked gels the stirring bar to swirl. Remove the flask and
should be discarded. occasionally swirl by hand until the mixture be-
Most types of gels can be cooked, poured, left comes less viscous once again, in about 1 min.
overnight, and run the following day. However, Return the flask to the stirrer and continue heat-
Tris-citrate/borate, Tris-citrate 111, lithium-bo- ing until boiling as above. This procedure takes
rate/Tris-citrate and Tris-HC1 gels tend to crack about 20 min.
during electrophoresis if used after this period of c. Cut the bottom out of a microwave oven having
storage. a stainless-steel interior in order to accommo-
Finally, there are a number of peculiarities date a magnetic stirring plate. While stirring,
associated with some gel buffers. Tris-borate- heat the starch-buffer mixture until it becomes
EDTA I1 gels tend to stick to the flask after cook- less viscous. Stop heating. (We have not used
ing. The problem can be overcome by lowering this method.)
the percentage of starch by 0.5-1 percent and/or
preparing an extra 20 ml of gel. Tris-citrate/bo- 5. Using an insulated glove, quickly transfer
rate and lithium-borate/Tris-citrate gels tend to molten gel to the aspiration shield, set flask on
split apart at the origin during running (see Pro- a heat pad and cover the open hole of the T-
tocol 4). Borate gels tend to be difficult to aspi- connector to apply vacuum for about 15 sec
rate; slight undercooking and/or vigorous shak- (Figures 5 and 7B). The mixture will resume
ing during aspiration reduce these problems (see boiling. Swirling of the flask may be required
also Protocol 4). during the first few seconds to avoid aspirat-
ing the gel out of the flask. Slowly release the
1. Locate a stable, horizontal surface to hold gel vacuum.
molds until gels are cool enough to move (=I
hr). The surface should be near the aspirator. 6. Rapidly pour the hot mixture into gel mold
filling evenly and almost overflowing (Figure
2. Prepare gel molds for receiving hot starch: 7C); avoid dribbles.
unglued wick molds (e.g., Micales et al., 1986)
must have the edges clamped; use masking 7. Immediately (within 1 min) remove any re-
tape and seal the open portions of the legs of maining air bubbles from the molten gel us-
wickless molds. Place molds on the table or ing a Pasteur pipette and pipette bulb.
bench on top of a paper towel. Label the pa- 8. Flush used flask in hot running water before
per towel (or masking tape) noting the type of remaining mixture solidifies.
gel buffer to be poured and the date. 9. After cooking all gels, and while they are
3. Weigh out 40 g (or appropriate weight) starch, cooling, fill buffer wells (trays).
place into a 1000-ml glass Erlenmeyer flask 10. Allow the gel to cool to ambient temperature,
(narrow mouth, heavy duty rim), and add about 45-60 min, and gently cover with plas-
400-ml gel buffer. Swirl contents until starch tic food wrap. With both hands, hold the
is well emulsified. wrap at one end. Allow the opposite free end
4. Cook gel using one of the following methods: to contact one edge of the gel. Slowly lower
Proteins: Isozyme Electrophoresis 81

Figure 7 (A) Cooking, (B) aspirating, and (C) pouring a starch gel.

the wrap allowing it to drop on the gel. If air ple wicks-rectangular pieces of filter paper
bubbles begin to form, lift the wrap and lower (Whatman No. 3) measuring 2-4 mm in width
it again; air bubbles induce malformations in and 1 mm taller than the gel mold. Wicks can be
the gel surface. Pulling/tugging of the wrap hand-cut or purchased. The following protocol is
should be avoided as this can split in the used for loading multiple gels, and for right-
forming gel matrix. Gently write the name of handed operators.
the gel buffer on the wrap using a felt-tip
marker. 1. Before loading, make sure that the buffer
wells have been filled and labeled.
11. Place gel in refrigerator for 1 hr, or allow to
continue to cool at ambient temperature for 2 2. If applicable, remove frozen homogenized
samples from freezer and initiate thawing,
and recentrifuge if desirable; keep thawed
samples chilled.
3. Number a piece of filter paper from 1 to the
Prcatocol3: Gel Lo3d-in.g number of samples being applied to a gel, in-
(Time: 10-20 min per gel) cluding tracking dye. Tape the -paper to the
The inoculation of protein extracts into horizontal table to the right of the operator.
gels is generally accomplished by the use of sam- 4. Make stacks of wicks on the numbered filter
82 Chapter 4 /Murphy, Sites, Buth & Haufler

paper. Each stack should contain as many

wicks as there are gels to be loaded.
5. Remove gels from refrigerator and fold the
plastic wrap back onto itself parallel to the
sample origin exposing half of the gel (or
6. Cut the edges of the gel free from the mold
using a microspatula.
7. Slowly cut gel origin vertically using a thin,
stainless steel microspatula and a gel origin
guide (Figures 4 and 8A). The guide must be
firmly held against the gel mold in order to
avoid slipping. Trial buffer gels should be cut
near the middle of the gel, others nearer to
one edge.
Thoroughly wet the first five stacks of wicks
with the first five tissue extracts, respectively,
using Pasteur pipettes or 1-200 pl pipetters.
Avoid cross-sample contamination by dispos-
ing of used pipette tips between samples.
Place the narrow side of the gel nearest to
operator. Gently open the origin of the well
about 5 mm by pushing the wide side of the
gel away. Using narrow-tip forceps, pick up
a damp wick from the first stack and place it
vertically into the gel origin against the nar-
row side, 1cm from the left side of the mold,
and in contact with the bottom of the gel Figure 8 (A) Cutting the gel origin using a gel origin
mold (Figure 8). Load the remaining four guide and (B) loading a starch gel.
samples, spacing the wicks about 1.5-2 mm
apart. Sequentially load any other gel(s).
Repeat steps 8 and 9 using the next series of 12. Recover gel with plastic wrap and perform
samples. Using tracking dye or a blank space electrophoresis as described below.
about every 10 specimens facilitates later gel
interpretation; allow 3 mm of space on either
side of a tracking dye wick. The last wick BrotdssoH 4: Electroplmoresis
should be soaked in tracking dye and located (Time: 4-24 hrs)
about 5 mm from the edge of the gel mold.
Two primary types of buffer systems are used:
Once all samples have-beenloaded, examine continuous and discontinuous. In continuous sys-
the wick placement from the bottom side of tems, the gel buffer is usually a 10%or less dilu-
the gel mold to be sure that all wicks are com- tion of the tray (electrode) buffer. In discontinuous
pletely inserted into the well. DO NOT shift systems (e.g., Tris-citrate/borate, and Tris-HC1) the
wicks laterally. Using a rolling action of the tray (borate tray buffer) and gel buffers are made
index finger remove any bubbles from the of different eledrolytes (Appendix 2); this system
bottom 'of the gel by pushing them to the has the effect of tightening isozyme bands during
sample origin. electrophoresis (see Richardson et al., 1986). The
Proteins: Isozyme Electrophoresis 83

tray buffer electrolytes can be observed to migrate without supervision). Splitting typically occurs
through the gel. when the tray buffer electrolytes pass through the
Certain kinds of gels have peculiarities, espe- origin. There are three remedies to this problem.
cially the discontinuous buffer systems. In many First, slightly overcook the gels during prepara-
buffer systems, such as Tris-HC1, the amperage tion. Second, push the gel halves together after
(electric current) drops as electrophoresis pro- the borate line has passed through the origin.
ceeds. Consequently, if running time is to be min- Third, following 1-2 hr of electrophoresis, wedge
imized the voltage should be progressively raised plastic drinking straws or thin glass rods between
to the maximum level (Table 5) about every half the gel and inside edge of gel mold thereby forc-
hour but without exceeding 75 mA. ing the gel halves together. These gels should be
Tris-citrate/borate, and lithium-borate/Tris- checked at the midpoint of electrophoresis to as-
citrate gels tend to split apart at the origin during sure that splitting has not occurred. Splits can be
electrophoresis, especially if the gels were cooked repaired by pushing the two halves of the gel
a day in advance or run overnight (i.e., run slowly back together.

Table 5
Recommended electrophoretic conditions for the wickless system described herein,
including electric potential in Vlcm and average duration
Buffer combination Vlcm Duration

Amine-citrate (morpholine) Ovemight 14 hr

Amine-citrate (propanol) Overnight 14 hr
Borate (continuous) Overnight 18 hr
6-7 hr
Borate (discontinuous) Overnight 14 hr
7-8 hr
6 7 hr
Overnight 20 hr
7-8 hr
Overnight 20 hr
10-12 hr
Overnight >18 hr
Overnight 18 hr
6 7 hr
12 hr
Ovemight >I4 hr
7 hr
Overnight 22-24 hr
Overnight 18 hr
5-6 hr
Overnight 12 hr
12 hr
5 hr
20 hr
18 hr
84 Chapter 4 / Murphy, Sites, Buth & Haufler

It is frequently possible to run gels much able to place a paper towel and glass plate be-
more rapidly than recommended, down to as lit- tween the gel and ice pack in order to prevent
tle as four hours. Because of the sieving effect of freezing of the gel surface.
starch gels, however, rapid running usually re- 4. Plug the well box top into the bottom, i.e.,
sults in less well-defined protein bands following connect the buffer well electrodes to the
staining. Moreover, as gels begin to heat up, resis- power supplies (Figure 9).
tance increases and further heating will likely oc-
5. Turn the power supply on, allow it to warm
cur-to the extent of melting gels!
up for a few minutes, and adjust to desired
1. If wickless gel molds are used, remove the voltage/amperage levels (Table 5).Amperage
masking tape from the legs. should not be allowed to exceed 100 mA, and
2. Place the gel mold in the buffer well box ori- preferably 75 mA, as overheating of the gel
enting the narrow end towards the cathode will likely occur.
(negative, black terminal). If wick molds are 6. After 15 min of electrophoresis, check track-
used, a sponge cloth must be used to com- ing dye by examining edge of gel mold to as-
plete the electric circuit between the gel and sure that the gel was properly oriented in the
buffer wells. While wearing rubber gloves, buffer well. If not, reverse polarity of the elec-
dip the sponge cloth into the well buffer and trodes at the power supply.
place it so that one end is in the buffer, and 7. Check ice levels every 2 hr if not running gels
one on the gel surface 1 cm onto the gel and in refrigeration.
under the plastic food wrap. 8. When tracking dye has reached the end of the -
3. Place either an aluminum tray filled with gel, turn power supply off and remove gel
crushed ice, or a frozen package of Blue Item (and gel mold) from buffer well box.
on the gel ensuring that the plastic wrap com-
pletely covers the gel and separates it from
the ice pack. If Blue Item is used, it is advis-
Protocol 5: Gel Slicing
(Time 5-10 min/ gel)
Once electrophoresis has been completed, the gels
Figure 9 Horizontal starch gel apparatus during
electrophoresis. The electrophoresis buffer tray is a need to be sliced and the slices placed in stain
slightly more complex version of that shown in Figure
3. The gel is being cooled by using Blue IcerM.
Proteins: Isozyme Elecfrophoresis 85

Figure 10 Gel slicing. (A) Use of a simple bow slicer use of a slicing tray. Note that when handling a gel
(see Figure 4). (B) A multiple slicer (plans available on slice, the fingers of both hands are touching to prevent
request). (C)Age1 sliced with a multiple slicer. (D) Gel stretching of the gel. Top glass (or plastic) plate in (A)
slice handling. The multiple slicer does not require the has been removed for clarity.

boxes. A number of methods have been devel- transfer the two parts separately. Improperly
oped including, among others, the use of bow cooked gels are difficult to handle by hand. Trans-
slicers and slicing trays (Figures 3 and IOA), mul- fer these slices by using plastic food wrap as a car-
tiple slicers (B.J.Turner, 1980; Figure 10B and C), rying medium.
and nylon string (thread) (Micales et al., 1986).Gel
slicing and handling should be carried out while 1. Using masking tape, label stain boxes with
wearing protective gloves, even though this in- the gel number, enzyme system or locus to be
creases the difficulty of the operations. stained, gel buffer, and date. (This step usu-
Several problems can occur during slicing, the ally is completed during electrophoresis.)
most common of which is that of splitting or tear- 2. Using a microspatula and gel origin guide, cut
ing the slices. Once a split has formed in a gel, it away the anodal and cathodal 1cm of the gel
can be extremely difficult to transfer slices from (or legs of the wickless gel), 3-5 mm of the
the tray to the stain box; splits usually result from edges of the gel, and notch the left anodal and
bending the gel too much while transferring it cathodal corners of the gel. Remove these
from one slicing tray to another. The easiest solu- edges and notched pieces from the mold, leav-
tion is to completely separate the split slice and ing the greater portion of the gel in the mold.
86 Chapter 4 / Murphy, Sites, Buth & Haufler

3. Separate halves at the origin and remove (Figure 10D) to the stain box. If an agar over-
wicks. The gel may be more difficult to move lay, UV fluorescing, or limited volume stain is
for some buffers (e.g., lithium-borate/Tris-cit- to be applied, then it is important that no bub-
rate). Using a paper towel, gently dry the top bles occur underneath the slice. Relatively ex-
of the gel, arrange the two pieces so that they pensive or critical stains should be made on
form a V separated by about 1 cm at one end, slices cut from the bottom of the gel.
and cover with a piece of plate glass or a slic- Repeat slicing. Always initiate subsequent
ing tray. slices from opposite ends of the gel to prevent
4. Invert the sandwiched gel and gently dry the uneven thinning. It may be necessary to re-
bottom of the gel. Choose the appropriate peat steps 4-5 if the remaining gel slides eas-
thickness of slicing tray (if applicable), center ily on the slicing tray. The top slice can be in-
it upside down on the bottom gel surface verted and used, although it is preferable to
with the tray ridges aligned with the origin, stain with an agar overlay. Remaining por-
and turn the gel right side up again. tions of gels can be temporarily saved (24+
5. Remove air bubbles between the gel and slic- hr) by wrapping.
ing surface. Failure to remove bubbles may
result in holes in the gel slice and/or render
the remaining gel incapable of being sliced. Protsscsl: 6: 14istc~chemicalStaii~iliilrrg
Re-cover top of the gel with second slicing (Time: 2 min to 6 hr/stain)
tray (or glass plate).
The distance of migration of specific proteins
6. Clean slicer wire with damp towel or steel through a starch gel is visualized by histochemi-
wool. cal staining. These stains (Appendix 1)consist of a
7. Orient the gel so that the apex of the V is fur- substrate on which a specific enzyme reacts, and a ,

thest away from the operator. Brace the (bot- detection mechanism such as a dye or substance
tom) slicing tray to prevent it from moving to- that fluoresces under long-wave (340 nm) UV. The
ward the operator during slicing. Place the common mechanisms for detection include (1)the
wire of the slicer on the raised ridges of the formation of a purple precipitate (formazan) by
slicing tray, press downward on the slicer, the reduction of NBT or MTT using PMS or DCIP
and in one continuous operation slowly as the intermediate electron carrier or reducer, re-
(about 3 cm per second) pull the wire through spectively; (2) the non-f1uo;escence of NAD,
the gel. Gels usually move toward the opera- whch is formed from fluorescent NADH; (3) flu-
tor slightly during slicing; do not stop pulling orescence of methylumbelliferone; (4) fast diazo
if this is observed, and DO NOT PRESS DOWN dye (e.g., esterases); and (5) the oxidized form of
O N THE TOP TRAY/PLATE. o-dianisidine diHCl producing an insoluble
8. Clean slicer wire with damp towel, or steel brown precipitate. Many stains also contain co-
wool. Do not immerse wire slicers in water. factors, coupling enzymes, and other requisite
Remove top tray/plate, carefully separate the molecules. Details of how each of these systems
gel from the bottom slice, and transfer the gel work are provided by Harris and Hopkinson
to the second slicing tray allowing for the V (1976), Richardson et al. (1986), and Manchenko
to have the opposite orientation (apex near (1994).A complete understanding of the concepts
operator). Similarly, move the anodal top slice is desirable but not absolutely necessary, although
but use both hands to support opposite sides such understanding greatly facilitates the resolu-
of the gel. Lift anodal top gel slice to second tion of staining problems when they occur.
tray, forming a V. Some stains (e.g., for PGM) are best applied
to the gels in the form of an agar overlay, or an
Open a plastic staining box and carefully agar-based gel containing stain components;
transfer the anodal and cathodal bottom slices agarose may be preferred over agar because the
Proteins: Isozyme Electrophoresis 87

latter inhibits the activity of some proteins be necessary (e.g., HADH). When mixing
through binding (Harris and Hopkinson, 1976). formazan-based stains, all powdered ingre-
Most laboratories use agar because it is much less dients should be dissolved in the stain buffer
expensive. The overlays serve the function of con- and pH adjustments should be made before
taining the precipitating dye, which prevents it adding cofactors, PMS, and NBT (or MTT).
from either diffusing over a broad area of the gel Once completely mixed, pour the stain onto
or becoming too diffuse to be observed. the gel and gently shake the box freeing the
Several UV-fluorescing stains (e.g., PGLU) gel from the bottom. Agar overlays are pre-
may be applied to the gel slices as filter paper pared by bringing a 0.7% (w/v) mixture of
overlays, the overlays being cut from Whatman agar/stain buffer to a boil, allowing it to set
1MM or other thin filter paper (Harris and Hop- until all agar grains have disappeared, cool-
kinson, 1976). However, we have not noticed an ing to just below 50°C, adding remaining
advantage over simply applying these stains di- staining components, and pouring onto the
rectly to the gel. gel slice. For the typical 50-ml stain, 35-40 ml
The quantity of specific chemicals in some of stain buffer is mixed with 0.35 g agar in a
recipes in Appendix 1varies from amounts speci- 125-ml Erlenmeyer flask; the remaining
fied in other sources (e.g., Selander et al., 1971). 10-15 ml of stain components are added to
These amounts are the minimum required to re- the warm agar just prior to covering the gel.
solve these protein systems from the maximum Under ideal conditions, the agar is prepared
diversity of taxa. Often these quantities can be re- in advance of slicing and staining by bring-
duced by applying less stain to a gel, especially ing the mixture to a boil in a microwave
once the region of activity has been identified. oven. The flask is corked or covered with
Most agar overlay stains can be easily accom- aluminum foil and kept in a 50°C water bath
plished using as little as 10 ml of stain solution. until used. Cooling of hot, molten agar can
Of the two dyes used in formazan-based be made rapid by the use of ice and an accu-
stains, MTT is cheaper, more toxic, and precipi- rate thermometer. The molten agar forms a
tates more rapidly than NBT but tends to diffuse gel at around 42°C. Some fluorescent stains
and is less stable. The two dyes can be used in are prepared as small agar overlays. Do not
concert. If NBT is yielding only faint bands ini- view the UV light or fluorescing gel without
tially, the addition of MTT during staining may the use of a UV light shield or protective
help to intensify the isozymes. glasses. Short wave lights are not necessary
For formazan stains, three components are and should not be used because of the addi-
particularly sensitive to light: PMS, MTT, and tional health hazard.
NBT. Therefore, the stock liquid solutions and 3. Most stains should be incubated at 37°C fol-
staining gel slices must be kept out of light. Stock lowing staining.
solutions should be stored in either amber glass
4. Staining gel slices must be continuously mon-
bottles and/or bottles wrapped in aluminum foil. itored to prevent overstaining, which results
All stains can be safely and conveniently pre-
in unresolvable, diffused, or smeared bands.
pared in Erlenmeyer flasks. Because some stains
Some stains must be scored and documented
contain liquid components only (e.g., LDH), these as soon as they are ready, sometimes within 5
may be mixed directly in the stain box so long as rnin of staining. Stains using insoluble precip-
the stain buffer is applied first. itates can be preserved (see below) and scored
1. Dry chemicals should be weighed and placed following the completion of all staining, even
in a 125-ml Erlenmeyer flask. on the following day.
2. Add the liquid components. Liquid compo- 5. If the stain has been applied as a liquid, and
nents can be handled safely using pipetting not an agar overlay, siphon off the stain solu-
devices. In some cases adjustment of pH will tion and save for appropriate hazardous
88 Chapter 4 / Murphy, Sites, Buth & Haufler
waste disposal. Completely cover the gel slice
with fixing solution (about 50 ml; Appendix applied as an agar overlay. Overstaining results
2) and refrigerate. If MTT is used as the dye, in very dense isozyme banding patterns. Occa-
do not flood the gel slice with fixative or the sionally, background "ghost bands" may be ob-
formazan dye will wash out of the gel; apply served. These bands result from the ability of an
only enough fixative to wet the gel slice enzyme to act on an alternative substrate (e.g.,
(about 20 ml). LDH acting on DL-glycericacid, the substrate of
GLYDH), presence of sufficient substrate in the
tissue extract, or contamination by bacteria,
Troubleshooting molds, and yeasts (e.g., ethanol and ADH). LDH,
A number of problems may be encountered fol- ADH and other isozymes can be identified either
lowing application of the stain, the most common by counterstaining, or by inclusion of the end
of which is the absence of enzyme activity on a product of the reaction, a procedure termed end-
gel. This may have several causes. (1) If the dura- product suppression. For example, pyruvic acid
tion of electrophoresis is too long or short, the en- suppresses (but does not stop) LDH, and pyra-
zymes may have migrated off of the gel or re- zole inhibits ADH. For some enzyme systems
mained in wicks in the origin, respectively. (2) (e.g., GLYDH), use of one or more suppressors is
It is possible that one (or more) of the stain com- required.
ponents were omitted from the stain recipe. Suc-
cessful staining may be possible by adding the
missing component to the stain. (3) Very weak ex- Protocol 7: Drying of Agar Overlays
pression typically results from too little of a given (Time: 6 hr)
component, or the use of a partially degraded so-
lution of coenzymes. Under these conditions, it Agar overlays can be dried on filter paper
will be necessary to add additional stain compo- and saved as documentation as follows:
nents, or reorder the coenzyme. If more than one
1. Cut filter paper (e.g., Whatman No. 1) to di-
stain is resolving inadequately, check for common
mensions allowing it to fit into a stain box
stain components, such as G6PDH. Coenzyme ac-
(12 x 17 cm) and label it with the enzyme sys-
tivity can be checked by electrophoresis and stain-
tem, gel number, and buffer conditions.
ing a small amount of the coenzyme along with
tissue extracts where activity has been previously 2. Decant or vacuum excess fixative from the
resolved. (4) A change in starch lot can result in stain box.
the necessity to change the conditions of elec- 3. Cut the agar free from the edges of the gel
trophoresis. (5) Shifts to a high pH can result in slice using a microspatula.
the conversion of NAD(P) to NAD(P)H. Check 4. Carefully overlay the filter paper on the agar
the pH of the final stain solution. (6) Finally, the overlay and then slowly lift the filter paper
addition of too much substrate or coenzyme can while separating the agar overlay from the gel
suppress enzyme activity. slice using a microspatula (Figure 11).
Smeared isozymes may result from use of the
5. Place the filter paper on a few paper towels
wrong electrophoresis buffer conditions, too high
agar-side up and allow to dry (severalhours).
a current (overheating), high concentrations of
lipids in the tissue extracts, or (rarely) improper 6. Once dry, curled overlays can be pressed flat
formation of the gel matrix. and wrapped in plastic for safe handling.
Diffuse isozymes can indicate overstaining, They should be stored in the dark and with
less than ideal electrophoresisconditions and/or light pressure to avoid recurling. BECAUSE THE
that an agar overlay stain should have been ap- AGAR WILL RETAIN DANGEROUS CHEMICALS FOR
plied. In the latter case, if light shalung of the gel YEARS, OVERLAYS SHOULD NEVER BE HANDLED
results in disturbance of the formazan precipitate WITHOUT WEARING PROTECTIVE GLOVES AND/OR
on top of the gel slice, then the stain should be UNLESS THEY ARE WRAPPED.
Proteins: Isozyme Electrophoresis 89

lated by choosing a tissue that will express the de-

sired gene products in the most scorable fashion.
The variables of subunit structure and genetic
control are &sassed below, followed by a brief in-
troduction to some common problems faced in the
interpretation of zymograms. For additional dis-
cussion see Harris and Hopkinson (1976), Rider and
Taylor (1980),Moss (1982),Richardson et al. (1986),
Weeden and Wendel (1989),and Buth (1990).
While many enzymes are monomeric pro-
teins (i.e., made up of one polypeptide chain) the
majority are multimeric (made of two or more
polypeptide chains), most often dimers and
Figure 11 Lifting an agar overlay from a gel slice.
tetramers. Harris and Hopkinson (1976), in a sur-
vey of the subunit structure of enzymes, found
P~atocol8:Documentation of Results 28% monomers, 43% dimers, 4% trimers, 24%
(Time: 1-5 min/slice) tetramers, and 1% octamers.
The simplest patterns of expression are single-
At the completion of staining, the isozyme pat- locus enzyme systems in diploid organisms. In a
terns should be documented by photography or homozygous individual, only a single allelic prod-
by drawing observed patterns on paper. Photog- uct is formed. Even if the enzyme in question is a =
raphy can be accomplished with a standard 35- multirner, only one kind of homogenous product .
mm camera, or more expensively with a Po- will be assembled; this product is a homomeric
laroidTM system. If 35-mm photography is used, a isozyme seen as a single zone of activity on a gel.
Y48 yellow filter should be fitted to the camera If the individual is heterozygous at this single lo-
lens in order to increase the contrast between the cus, two kinds of allelic products are formed. In
stained isozyme patterns and the gel background; the case of monomeric enzymes, the two allelic
this filter is necessary for documenting W stains. products are produced in equal quantity, do not
Photography is best carried out on a copy stand interact strzlcturdy and are expressed equally in a
fitted with a light box. A polarizing filter helps to given tissue of an individual (1:l ratio). A zymo-
cut glare if the gel slice is illuminated from above gram illustrating triallelic variation involving a
but it should not be used when photographing monomeric enzyme is shown in Figure 12. In the
U V stains. If using a UV lamp rather than a trans-
illuminator, locate it close to the gel. When pho-
tographing, we place the stain box label on the gel
to document each photograph.

0 ~ 1 2. 3 4 . 5 6 7
The interpretation of the band patterns comprising Figure 12 Photograph exhibiting triallelic variation
the zymogram requires the knowledge of the sub- at the phosphoglucomutase locus (Pgm-A)in muscle
unit structure and the genetic control of the en- extracts from the cyprinid fish L u x i l u s cardinalis.
zyme system. As discussed in the gene expression Specimens 1, 2, and 4 are homozygous expressing
only the 82 homomer; specimen 3 is heterozygous
section, the tissue examined for enzyme activity expressing both 82 and 100 homomers; specimens 5,6,
may limit the number of gene products or sub- and 7 are also heterozygous expressing both 68 and 82
units expressed. These variables may be manipu- homomers.
90 Chapter 4 / Murphy, Sites, Buth & Haufler

case of multimeric enzymes, the two allelic prod- Hardy-Weinberg expectations for the distribution
ucts are also produced in equal quantity in a given of allozyme products of a given locus is usually a
tissue but the products will usually randomly as- safe one. Violation of this assumption suggests
semble to form all expected heteromers, in addi- that additional study is necessary, beginning with
tion to homomers. It is usually the case that the a reassessment of the scoring of that enzyme sys-
subunits of multimeric enzymes form homomers tem. Scoring only clear bands and omitting
and heteromers at random, yielding banding pat- smeared zones may overestimate the frequency of
terns in predictable ratios. Because heteromers of homozygotes. Frequently the report of 50% allele
similar composition can be assembled in several 1 and 50% allele 2 for a given locus in a table of al-
ways, the ratio of expected intensity of enzyme ac- lele frequencies is the result of incorrect scoring of
tivity differs among isozymes according to the an entire sample (n > 5) as heterozygotes.
subunit structure of the enzyme (Figure 13). This Difficulty in scoring gels can occur when any
variation is detailed in Table 6. of the other assumptions discussed previously are
The situation is more complex for multilocus violated. Exceptions to expected subunit interac-
enzyme systems. Multimeric gene products of tions and genetic control are often encountered.
multiple loci in an enzyme system often retain The random association of subunits of multimeric
their ability to form heteromers, and the number enzymes sometimes is restricted, yielding fewer
of isozymes formed can be considerable where zones of activity than expected. For example, cre-
heterozygosity occurs. Harris and Hopkinson atine kinase is a dimer in all vertebrates but the
(1976) provided the following equation for the

- -
computation of the expected number of isozymes
(i) under these circumstances:
Homozygote Heterozygote Homozygote

where L = the total number of loci, h = the num-

I. 1

ber of heterozygous loci, and n = the number of

subunits for this enzyme. The multilocus situation +I 1-
differs from its single-locus counterpart in that, Dimer
whereas allelic products of a single locus can ac-
count for equal quantities of both products, the
products of two different loci would rarely con-

tribute equal quantities of both products in the
same tissue. The predictable symmetrical ratios of
isozymes in heterozygotes cannot be extended to
the multiple loci unless, by chance, the two gene
I- 3

products are produced in equivalent proportions.
Examination of enzyme expression in multiple
tissues may aid in distinguishing single-locus
heterozygosity from similar isozyme patterns re- Tetramer
+I -4

sulting from interactive multilocus products. 1

However, the question of a two-allele, single-lo-
cus model versus that of interactive products of
two homozygous loci can also be addressed by Figure 13 Diagram of isozyme patterns expected in
homozygotes and heterozygotes for enzymes of com-
comparing the frequency of heterozygotes to the mon subunit composition. Modified from Harris and
predictions of Hardy-Weinberg equilibrium (e.g., Hopkinson (1976). Ratios of intensity of isozyme activ-
Ferris and Whitt, 1978a). The assumption of ity in heterozygotes are indicated. See Table 6.
Proteins: Isozyme Electrophoresis 91

Table 6
Subunit structures of homomeric and heteromeric isozymes in heterozygotesa
Monomer Dimer Trimer Tetramer

Homomer 1

Homomer 2
aModified from Harris and Hopkinson (1976). Two alleles at this single locus determine polypeptide units
1 and 2 respectively. Random combination of subunits of mulimeric enzymes is assumed.

heterodimer is not formed in heterozygotes at the ity or homozygosity should be correlated among
Ck-A locus in teleost fishes (Ferris and Whitt, tissues of an individual (e.g., Murphy and Crab-
1978b).The subunit structure of enzymes often is tree, 1983).The probability for unlinked multiple
quite conservative across taxa; however, some en- loci to covary in such a way can be addressed sta-
zymes have been reported to have a variety of tistically (see Hart1 and Clark, 1989).
structures in different groups of organisms In all studies that deal with questions of
(Manchenko, 1988). These reports may reflect ei- whether mobilities of electromorphs are equiva-
ther real structural differences among taxa or the lent or whether an individual is heterozygous at
restriction of heteromer formation misinterpreted a locus, the resolution of discrete zones of enzyme
as structural differences. Rigorous testing (e.g., activity on a gel is essential. If multiple buffer sys-
Ferris and Whitt, 1978b) should be applied in tems are not used or if tissue extracts no longer
these cases. On rare occasions, allelic products provide sufficient enzyme activity, the resolution
have different catalytic properties and expected may be inadequate. Interpretation of these subop-
ratios of isozyme expression are not realized. Ex- timal gels results in dubious data sets. For exarn-
amination of a large series of individuals that re- ple, overstaining will obscure the subtle differ-
solve all heterozygous and homozygous cate- ences in relative activity of isozymes. In spite of
gories should allow the correct interpretation of the resolution of discrete zones of enzyme activ-
such variation. Epigenetic effects yield isozymes ity and efforts to limit enzyme expression to pri-
of different electrophoretic mobilities in different mary isozymes, some non-genetic subbanding
tissues and can suggest the action of more struc- may confound the interpretation of gels. The pro-
tural loci than are actually present. If only a single duction of these secondary isozymes, or sub-
locus is active in this case, apparent heterozygos- bands, may vary by tissue location and age, en-
92 Chapter 4 /Murphy, Sites, Buth & Haufler

+ I Gpi-A products

1 2 3 4 5 6 7 8 9 1 0
Gpi-A genotypes: 125 125 125 125 125 125 100 100 120 100
125 125 125 125 125 100 100 100 100 100

Gpi-B genotypes: - 100 -

28 - 100 - 100 -
100 - 100 143 100 100 164
100 100 100 100 100 100 100 100 100 100
Figure 14 Photograph exhibiting variation at glu- nalis (lanes 1-6) and Luxilus zonatus (lanes 7-10).
cose-6-phosphate isomerase loci Gpi-A and Gpi-B in Genotypes are listed for each locus. Notice the sub-
muscle extracts from the cyprinid fishes Luxilus cardi- banding.



(A) (B) (C)
Figure 15 Photograph demonstrating a gel buffer in no case are all of the anticipated five isozymes
screen from rattlesnakes for the enzyme system L-lac- resolved (see Figures 13 and 16). Buffer (A) suppresses
tate dehydrogenase. (A) Tris-citrate I11 pH 7.0. (B) Tris- the activity of the more anionic system, products of
citrate/borate pH 8.2. (C) Tris-citrate I1 pH 8.0. (D) the heart-predominating Ldh-B locus. Isozymes of the
Tris-citrate-EDTA pH 7.0. (E) Phosphate-citratepH 7.0. slower skeletal-muscle-predominating system Ldh-A
Increasing the pH also increases the net charge and cannot be resolved adequately on systems (D) and (El.
relative mobility of the isozymes. Three or four Note that the minislices are uniquely notched.
isozymes are observed, depending on the buffer, but
Proteins: Isozyrne Electrophoresis 93

Anode erodimers in relevant individuals can provide an

additional hint of heterozygosity in this example
(e.g., specimens 6 and 9 in Figure 14 in which the
variation at the Gpi-A locus is subtle).
Having noted the basics of gel interpretation
we provide a few additional examples. The effect
of using different buffers to resolve enzyme vari-
ability is shown in Figure 15. Alternative buffers
can differentially affect both relative mobility and
activity of isozymes. Although not shown, some
buffers would result in smeared isozyrne patterns.
Figure 16 demonstrates that multiple buffers may
be required to resolve allelic variants at different
loci of the same enzyme system. Diagnosis of a
posttranslational modification is shown in Figure
1 2 3 4 5 6 7 8 Cathode 17, although the exact nature of this modification
Figure 16 Photograph demonstrating intra- and is unknown.
interlocus variability of tetrameric L-lactate dehydro- Some enzyme systems may appear as back-
genase (LDH) isozymes in spring peeper frogs, Hyla ground upon staining for others. These may be
(Pseudacris) crucifer. The more anodal system is the
heart-predominating locus, Ldh-B, and the cationic
locus (on this buffer system) is muscle-predominating
Ldh-A. Here resolution is inadequate for interpreting
variation at the Ldh-A locus but is excellent for Ldh-B;
the buffer revealing Ldh-A variation masks that of Ldh-B.
Specimens 1, 2, and 5 are homozygous at both loci
although 1 has a different allele expressed at Ldh-A;
these specimens show the five expected isozymes
(Figure 13). Lanes 3,4, and 8 are heterozygous at Ldh-A
but homozygous for two different alleles at Ldh-B. sMDH
Lanes 6 and 7 are heterozygous at both loci.

zyme system, and electrophoresis buffer used. - Origin

Resolution of this problem often comes by chang- mMDH
ing to another buffer or resolving variation at the
locus. The subbanding problem is illustrated for
dimeric glucose-6-phosphate isomerase (GPI) in PTM
Figure 14. This enzyme system is controlled by
two loci, now known as Gpi-A and Gpi-B, in
teleost fishes (Avise and Kitto, 1973); products of Figure 17 Gel of malate dehydrogenase (MDH) from
the latter locus predominate in muscle tissue and salamanders (Ambystoma maculatum) showing varia-
an interlocus heterodimer is usually formed. tion in anionic isozymes (the supernatant locus sMdh-
A) and cationic isozymes (the mitochondria1 locus
Isozymes of Gpi-B often yield two anodal sub- mMdh-A), and a posttranslation modification. sMdh-A
bands beyond each homomer or heteromer. This is dimeric and heterozygotes appear similar to those
pattern might be confused with that of heterozy- of GPI (Figure 14). mMdh-A products are tetrameric
gotes. However, as Figure 14 illustrates, heterozy- and heterozygotes near the gel origin (arrow) appear
gotes serve to clanfy the situation even if their ho- smeared because the intralocus heteropolymers are
too close together to be resolved. A posttranslation
momers and heteromers are superimposed on modification (PTM) of mMdh-A products results in
some subbands and obscure the expected ratios of more highly charged isozymes which in this case lack
isozymes. The presence of two interlocus het- the intralocus heteropolymers.
94 Chapter 4 1Murphy, Sites, Buth €3 Haufler


4 Origin
Figure 18 Photograph showing the resolution of
superoxide dismutase (SOD) isozymes (light bands) as
background on a gel stained for glycero~3-phosphate
dehydrogenase (G3PDH) in spring peeper frogs, Hyla terpretation of the zyrnograms. Documentation of
(Pseudacris) crucifer. results through the publication of either gel
photographs or zymograms is recommended
desirable, as in the case of observing superoxide
dismutase (SOD) following staining for glycerol-
3-phosphate dehydrogenase (G3PDH; Figure 18), ENZYME AND LOCUS
or undesirable (Figure 19). NONIENCLATURE
Figure 20 documents the necessity of choos-
ing the correct array of tissues to be surveyed. Fi- The effective communication of data derived from
nally, Figure 21 shows unacceptable resolution of protein electrophoresisis critical to a clear under-
an isozyme system. Optimal resolution of en- standing of any study. Consequently, there is a
zyme activity will facilitate a correct genetic in- need for a reasonably standard.system of enzyme



1 2 3
Figure 19 Photograph showing extensive variability which may be misinterpreted as a second MPI locus.
in mannose-6-phosphate isomerase (MPI) isozymes All individuals except those indicated by arrows are
among some hylid frogs along with background reso- Hyla (Pseudacris) crucifer. The more anodal isozymes in
lution of L-lactate dehydrogenase products (LDH), species 2, H. cadaverina, are Ldh-B products.
Proteins: Isozyme Electrophoresis 95

*+- -\*
, 7
",.'" -P*;aP locus of vertebrates is referred to as Ldh-B, and the
locus predominating in skeletal muscle Ldh-A
i 1 - Ck-Cproducts
(Fisher et al., 1980). Where the locus homologies
have not been specifically analyzed by immuno-
6%' '4<1 - AC heterodimers
logical affinity, or other means, we refer to them
* . by number (e.g., Est-1, Gp-1). Duplicated paralo-
- Ck-A products
gous loci, for example the duplicated G3pdh-A lo-
- Ak-A products cus of some squamate reptiles (Sites and Murphy,
19911, are noted by following the locus designa-
tion with a number: G3pdh-Al, and G3pdh-A2. Fi-
1 2 3 1 2 3 1 2 3 1 2 3
%-'-V'-+w nally, many loci are expressed in subcellular or-
Muscle Heart Liver Stomach ganelles and specific designations have been
Figure 20 Creatine kinase (CK) isozymes from the proposed for them in the form of a lower case pre-
marine toad, Bufo marinus, showing differences in tis- fix: among animals, m = mitochondrial loci (e.g.,
sue expression and the presence of interlocus het- mAat-A); s = cytosolic (soluble or supernatant;
eromers. In stomach, only products of the Ck-C locus when enzymes systems are expressed by dupli-
are expressed whereas in skeletal muscle only Ck-A cate loci and specialized to function at the subcel-
is seen. In heart tissue, both locus products are
expressed, albeit weakly, and the interlocus hetero- lular level); 1 = lysosomal; p = peroxisomal; and
polymer (heterodimer) is present; the pure locus prod- among plants, s = cytosol; mt = mitochondrial; p =
ucts (homodimers) are not expressed in equal intensi- plastid; mb = microbody; c = cell wall. The suffix
ty, eliminating the possibility of a heterozygotic state. designations of Shaklee et al. (1992),which denote
CK is not expressed in liver. Adenylate kinase (Ak-A
locus) activity is also resolved and limited to skeletal
regulatory loci (r; e.g., Ldh-Ar) and pseudogenes -1
muscle tissue. (p = documented; 1 = ambiguous orthology), also
can be incorporated into our system.
Specific alleles at each locus are denoted with
and locus nomenclature. Shaklee et al. (1992) rec- parentheses, referred to by lower case letters, and
ognized this need and recently proposed a system follow the locus abbreviation; thus the a allele of
for fish based on human gene nomenclature. Ldh-A is accordingly designated as Ldh-A(a). The
However, we believe this system can be simpli- genotype of a specific homozygous individual
fied by eliminating asterisks and comma nota- having allele a is referred to as Ldh-A(a/a). Simi-
tions. We also prefer not to use italics to designate larly, a heterozygous individual having alleles a
loci, although many publications (such as this and b would be referred to as Ldh-A(a/b). Simi-
book) may require their use to distinguish them
from other text.
Our nomenclature generally follows Buth
(1983,1984b), Murphy and Crabtree (1983), and
Shaklee et al. (1992) with some modifications.
Enzyme systems are referred to by upper case let-
ters; for example, L-lactate dehydrogenase is re-
ferred to as LDH. (Other recommended abbrevia-
tions are given in Appendix 1).In some enzyme
systems, the specific locus homologies (ortholo-
gies) have been identified by tissue-specificdistri- Figure 21 Unacceptable resolution of pyruvate kinase
butions, immunological affinities, physiological (PK) in spring peeper frogs, Hyla (Pseudacris) crucifer.
properties, and relative mobilities. Specific loci are Smeared bands could result from using an incorrect
buffer system, overstaining, a bad gel, or bad samples
noted using system identification but with lower (too-high lipid content, denatured proteins, old sam-
case letters except for the first letter which is up- ples, etc.). In addition, the sample wicks have not been
per case. For example, the heart-predominating spaced adequately.
96 Chapter 4 /Murphy, Sites, Buth & Haufler

larly, numbered alleles should be separated by a sources usually extends only to the secondary lit-
forward slash, as in Ldh-A(100/125). erature wherein modifications are already noted.
Particular interlocus isozymes resulting from With few exceptions, the enzyme names and
subunit interactions in multilocus systems are Enzyme Commission (EC) numbers used in this
designated using enzyme system notation (capi- compilation are those recommended by the Inter-
tal letters) followed by subscripts that designate national Union of Biochemistry (IUBNC, 1984).
subunits, e.g., LDH-A3Bl, or simply abbreviated Abbreviations of enzyme names are placed in
A3B1. In heterozygous individuals, polymeric en- capital letters; abbreviations are developed from
zymes (enzymes composed of multiple subunits) the IUBNC (1984) recommended names and
yield multiple isozymes. Intralocus polymeric sometimes differ from common usage by the ad-
isozymes, formed from the interactions of differ- dition of letters for clarity. The listing of named
ent alleles at a particular locus in a heterozygote, loci controlling each enzyme system is beyond the
are denoted by locus designation with allelic sub- scope of this appendix and other abbreviations
script notation showing the number of con- are defined in the glossary.
stituent allelic subunits, e.g., Ldh-A(a3bl),or sSod- The quaternary structures for enzymes listed
A2(albl). [Note that in all allozyme studies, a herein were taken from those reported by Harris
heterozygous condition is simply denoted as Ldh- and Hopkinson (1976), D.E. Soltis et al. (1983),
A(a/b) or sSod-A2(a/b).lFinally, in polymeric, mul- Richardson et al. (1986), Aebersold et al. (1987),
tilocus systems, it is possible for subunits pro- Manchenko (1988),and personal communications
duced by different loci (e.g., A or B) and from a number of researchers. For some enzymes,
alternative alleles at a particular locus [e.g., A(a) these structures are well documented and conser- -

or A(b)] to combine within a single tissue yield- vative across taxa. For others (e.g., catalase and
ing a multitude of distinguishable isozymes [see glucose-6-phosphate dehydrogenase), several
Gorman and Shochat (1972) for the resolution of quaternary structures have been reported '

15 LDH isozyrnes.] For this we suggest combin- (Manchenko, 1988). Whether these and other en-
ing system and allelic notation as follows: LDH- zymes actually exist in multiple structural forms
A3(a2bl)B,where two subunits of Ldh-A(a), and or have a conserved single multimeric structure
one subunit each of A(b) and B(a), combine to that is expressed as restricted subunit combina-
form a single LDH isozyme. tions remains to be investigated.
Many of the biochemicals used in enzyme
stains are marketed in a number of forms. In some
APPENDIX 1: ENZYME S'TAINING cases, ultrapurity is not required and considerable
FORMULAS savings can be achieved through the purchaseof a
lesser grade. In some cases, the choice of a partic-
(Compiled by Donald G. Buth and Robert W. ular salt may be critical. We have listed (Table 4)
Murphy) the product number of many of these stain com-
ponents keyed to the catalog of the Sigma Chemi-
Formulas for enzyme stains frequently are modi- cal Company (P.O. Box 14508, St. Louis, Missouri
fied and republished, often as compilations for 63178 U.S.A.) to allow the reader to evaluate the
specific groups of organisms or even for single kind and cost of these biochemicals. This choice
species. Textbook treatments often provide a lim- does not necessarily represent our endorsement of
ited introduction to the vast array of stains avail- these products.
able, whereas listings for specific groups of or- Most of the stains below are based on a stan-
ganisms often are limited to those systems well dard volume of 50 ml suitable for gel slices from
known or expressed only in those groups. Our most horizontal starch gel apparatus (scaled down
listing is not meant to be all-inclusive; our selec- from stain formulas for 100-ml volume used com-
tion is biased toward economical systems in use monly for the macroscale vertical apparatus of ear-
by botanists and zoologists. Our reference to stain lier studies). Some investigators have reduced the
Proteins: Isozyme Electrophoresis 97

staining solution volume even further to conserve Dissolve the substrate:

expensive stain components; many of these reduc-
tions are noted and others may be warranted.
galactosamide 5 mg
The agar overlay method (see text) is recom-
dimethyl sulfoxide 0.25 ml
mended for most stains by some (e.g., Shaklee
Add the dissolved substrate to:
and Keenan, 1986). For 50-ml stains, we recom-
mend 35-40 ml of buffer with the agar, and 10-15 0.1 M phosphate-citrate buffer, pH 9.5 15 ml
ml buffer with the stain components. Specific rec-
ommendations are made for stains using reduced Incubate, then view under UV light (long wave-
volumes. See text for specific instructions and length). Zones of activity will appear as bright ar-
suggestions. Our staining methods reflect our eas. To enhance fluorescence, spray the gel with a
own biases in choice of biochemical reagents and concentrated solution of ammonium hydroxide.
thiir means of storage and application. For exam- This stain was described by Aebersold et al. (1987).
ple, we see no need to use the more expensive
NADP in stains requiring glucose-6-phosphate
dehydrogenase when an NAD-dependent form of
G6PDH can be used (Buth and Murphy, 1980).
NADP is used in solid form herein [see malate de- Dimer This enzyme formerly was called hex-
hydrogenase NADP (MDHP)] but may be suit- osaminidase (HA). This stain may be prepared as
able in liquid stock for many other stains. We pre- an agar overlay (0.17 g agar, 10 ml H20) which is
fer NBT to MTT (see text). Other individual added to the substrate below, or simply pour the
preferences abound; for instance, Ayala et al. following onto the gel surface:
(1972) recommended adding phenazine metho-
sulfate to stains after an hour or more of incuba- glucosaminide
tion with the other components. We urge re- 5 mg
dimethyl sulfoxide 0.25 ml
searchers to experiment with the options listed
among these recipes and to try other modifica- Add the dissolved substrate to:
tions of their own invention.
0.1 M phosphate-citrate buffer, pH 4.5 15 ml
Unless otherwise indicated, the stained gel
slices should be incubated in the dark at 37°C. Incubate, then view under UV light (long wave-
Recipes for stain fixing solutions follow the stain length). Zones of activity will appear as bright ar-
recipes. eas. To enhance fluorescence, spray the gel slice
The costs of most stains listed below are pro- with a concentrated solution of ammonium hy-
vided in Rainboth and Buth (1992) and are based droxide. This stain was described by Aebersold et
on the 1992 Sigma Chemical Company catalog. al. (1987).
The relative costs in U.S. dollars are provided
with each stain in the following notation: $,
<$1.00; $$, $1.00-$2.00; $$$, $2.01-$5.00; $$$$, Acid Phsssphatase QACP)$
>$5.00. (EC3.1,3,2)
Monomer or Dimer Two stains are required in
many vertebrates in order to resolve both the red-
cell and tissue ACP isozymes. For dimeric tissue
isozymes incubate the gel slice at ambient tem-
perature for 30 min in a 0.05 M sodium acetate
Dimer Prepare this stain as an agar overlay (0.17 buffer, pH 6.0 (= 0.33 g sodium acetate in 50 ml
g agar in 10 ml water and combine with substrate H 2 0 with a minor pH adjustment). Drain, then
mixture). add the following solution to the gel slice:
98 Chapter 4 / Murphy, Sites, Buth & Haujler

sodium acetate (NaC2H3O2-3H20) 0.33 g This stain was modified from Harris and Hopkin-
a-naphthyl acid phosphate 0.15 g son (1976) and Siciliano and Shaw (1976). Note
fast garnet GBC 0.03 g that the magnesium chloride used is 1.0M, not 0.1
H20 50 ml M as is used in many other enzyme stains. Cis-
aconitic acid stock solutions should be made in
The pH of the staining solution is about 5.5 so fur- small quantities as it seems to decompose in 1-2
ther adjustment is usually unnecessary; the pH months.
should be 5.0-6.0. The stain was modified from
Shaw and Prasad (1970). Werth (1985) recom-
mended the use of a stock solution of the substrate Adenosine Deaminase (ADA) $$
Pnapththyl acid phosphate in 70% acetone (use 1 (EC 3.5,4,4)
ml of a 1% solution). D.E. Soltis et al. (1983) and
Werth (1985) recommended the use of fast garnet Monomer This stain may be prepared as an agar
GBC salt as a substitute for black K salt. Sigma fast overlay.
black K salt has not provided satisfactory results 0.2 M Tris-HC1, pH 8.0
in studies of reptiles. This stain was modified from H20
Harris and Hopkinson (1976) and does not resolve adenosine
red-cell ACP in many vertebrates, including many arsenic acid
reptiles. Monomeric red-cell ACP isozymes, also xanthine oxidase
known as erythrocytic acid phosphatase (EAP; $$),
nucleoside phosphorylase
may be stained as follows:
5 mg/ml MTT
0.05 M citrate buffer, pH 6.0 50 ml 5 mg/ml PMS
phenolphthalein diphosphate 0.2 g
This stain was modified from Spencer et al. (1968).
Incubate for 1 hr, decant the staining solution and
spray the gel surface with a concentrated solution
of ammonium hydroxide. Zones of activity will ap-
pear as pink bands. This same stain was described
by Harris and Hopkinson (1976) who recom-
Monomer This stain may be prepared as an agar
mended 4 hr of incubation at 37OC. In some verte-
brates tissue ACP isozymes can also be resolved.
0.2 M Tris-HC1, pH 8.0 50 ml
0.1 M MgC12 6 ml
ad6nosine 5'-diphosphate 0.03 g
D(+)glucose 0.1 g
Monomer This enzyme was known formerly as hexokinase 200 u
aconitase (ACO or ACON). Mitochondria1 and su- G6PDH 40 NAD U
pernatant/cytosolic forms are known (Harris and 10 mg/ml NAD 2 ml
Hopkinson, 1976). This stain may be prepared as 5 mg/ml NBT 1 ml
an agar overlay. 5 mg/ml PMS 1 ml
0.2 M Tris-HC1, pH 8.0 50 ml This stain was described by Buth and Murphy
1.0 M MgC12 (see note below) 1.5 ml (1980) as modified from Fildes and Harris (1966).
0.1 M cis-aconitic acid, pH 8.0 5ml A more sensitive, but more expensive, fluorescent
isocitric dehydrogenase 3U stain modified from Harris and Hopkinson (1976)
NADP 0.01 g may also be used.
5 mg/ml MTT 1 ml
5 mg/ml PMS 1 rnl
Proteins: lsozyrne Electrophoresis 99

Alanine Amins t ransf erase 4Ai,A17) 6 0.2 M Tris-HC1, pH 8.0

(EC 2,6.2,2) 95 or 100%ethanol
10 mg/ml NAD
Dimer This enzyme was known formerly as glu- 5 mg/ml NBT
tarnic-pyruvic transaminase (GPT).Prepare as an
5 mg/ml PMS
agar overlay (0.07 g agar in 5 ml buffer and then
combine with the following): Products of some ADH loci may be resolved bet-
ter using other alcohols (e.g., 0.2 ml 98% l-hexa-
0.2 M Tris-HC1, pH 7.0 5 ml
nol, or 0.3 ml 1-amyl alcohol). Some related en-
DL-alanine 0.04 g
zymes are named specifically (e.g., octanol
a-ketoglutaric acid 0.02 g dehydrogenase: EC although the sub-
NADH 0.02 g strate, octanol, also may resolve other alcohols
L-lacticdehydrogenase 25 U (e.g., ADH). This stain was modified from Brewer
Monitor the development of expression under UV (1970). Werth (1985) warned of fume contamina-
light (long wavelength). Enzyme activity is indi- tion of nearby incubating gels and recommended
cated by zones of defluorescence. This stain was sealing the ADH stain box with plastic food wrap.
modified from Harris and Hopkinson (1976) and
Siciliano and Shaw (1976). See also Casillas et al. A.%kalinePhssphatase (ALP) $;
(1982). An alternative stain was described by Ae-
bersold et al. (1987).
(EC 3.1.3s3.)
Monomer or Dimer First combine the following:
0.2 M Tris-HC1, pH 9.0
Monomer Prepare as an agar overlay (0.07 g agar
in 5 ml buffer). Then add:
0.2 M Tris-HC1, pH 8.0 a-napthyl acid phosphate 0.15 g
pyruvic acid polyvinylpyrrolidone (PVP) 0.15 g
L-alanine fast blue RR salt 0.05 g
NADH This stain was modified from Boyer (1961) and
Incubate and view under UV light (long wave- Ayala et al. (1972).
length). Enzyme activity is indicated by zones of
defluorescence. This stain was modified from a-1.-Arabinofuranosidase (ARAB) $$
Shaklee and Keenan (1986). See also Dando et al.
(1981); this enzyme may be limited to certain in- 1E.C 3,2,2,55b
vertebrate animals (Manchenko, 1988). Subunit structure unknown.
0.1 M phosphate-citrate buffer, pH 4.0 5 ml
Alcohol Dehydrogenase (ADHI $ 4-methylumbelliferyl-L-arabinoside 0.01 g
(ECl e l ~ l . l ) Incubate for =30 min and then view under UV
Dimer Mix the substrate alcohol and the buffer light (long wavelength). Zones of activity will ap-
thoroughly prior to adding the other stain com- pear as bright areas. To enhance fluorescence,
ponents. An agar overlay may be appropriate in spray the gel slice with a concentrated ammo-
some circumstances. nium hydroxide solution. T h s stain was modified
from Harris and Hopkinson (1976).
100 Chapter 4 / Murphy, Sites, Buth & Haufler

Argixaine Kinase (ARK) $$$$ This stain was provided by J.P. Bogart. An alter-
(EC 2-7.3.3) native stain modified from M.K. Schwartz et al.
(1963) was given in the first edition of Molecular
Dimer The following stain may also yield adeny- Systematics. A final pH of 8.0 is critical to the suc-
late kinase (AK) gene products. A control slice cess of this stain (D.E. Soltis et al., 1983).A more
from the same gel must be stained specifically for sensitive, but more expensive, fluorescent stain is
AK to ascertain, by a process of elimination, found in Harris and Hopkinson (1976).
which zones of activity are ARK. Prepared as an
agar overlay (0.09 g agar in 8 ml buffer).
Calcium-Binding Protein.5 (CB.P) $$
0.2 M Tris-HC1, pH 7.0 12 ml
0.1 M MgC12 1 ml [nhsn-sped$icl
adenosine 5'-diphosphate 0.02 g Monomer CBPs migrate rapidly toward the anode
glucose 0.04 g and are somewhat diffuse in appearance (Buth,
hexokinase 200 u 1979, 1982b). The creatine kinase gene products
phospho-L-arginine 0.02 g (Ck-A) and other proteins that predominate in the
G6PDH 40 NAD U tissue examined (e.g., those often scored as gen-
10 mg/ml NAD 1ml eral proteins) will also stain via this procedure.
5 mg/ml NBT 1ml Dissolve the dye in water:
5 mg/ml PMS 1 rnl
This stain was modified from Shaklee and Keenan brilliant blue G dye
(1986). This enzyme is present only in some in-
Then add acids:
vertebrate animals (Shaklee and Keenan, 1986).
trichloroacetic acid 7.5 g
5-sulfosalicylic acid 2.5 g
Aspartate Paminotransferrase (AAT) $
dEC This stain was modified from Massaro and Mark-
ert (1968).
Dimer This enzyme was known formerly as glu-
tamate-oxaloacetate transaminase (GOT). Mito-
chondrial and supernatant/cytosolic forms are
known (Harris and Hopkinson, 1976). This en-
zyme is best resolved from extracts of relatively
Tetramer? Allow the gel slice to warm to ambient
fresh tissues.
temperature, then add:
Substrate solution:
0.06 M sodium thiosulfate 15 ml
ddH20 400 ml
3% hydrogen peroxide 35 ml
L-asparticacid 1.33 g
a-ketoglutaric acid 0.365 g Incubate at ambient temperature for 1 min, drain,
polyvinylpyrrolidone 2.5 g and add:
Na2EDTA 0.5 g 0.09 M potassium iodide (KI) 50 ml
Na2HP04 14.2 g
Flush the gel slice quickly with water to remove
Adjust final solution to 500 ml. Minor pH adjust- KI as soon as the white zones of catalase activity
ment to 8.0 with 4.0 M NaOH might be required. are visible against the blue background. DO NOT
Keep refrigerated. For one stain add: place stained gel slice in gel fixitive! Photograph
the developed gel slice immediately if possible.
0.2 M Tris/HCl, pH 8.0
substrate solution The gel slice may be stored in water in a dark re-
frigerator for a few days but the blue back-
fast blue BB salt
Proteins: Isozyme Electrophoresis 101

ground stain will be lost eventually. This stain would be to consider this enzyme under the cate-
was modified from Brewer (1970). Siciliano and gory of generic aminopeptidases EC 3.4.-.-.
Shaw (1976) recommended a longer incubation
Incubate the gel slice in the following solution
period (15 min) for the initial solution. D.E. Soltis
for 30-60 min:
et al. (1983) and Werth (1985) noted that up to 1
ml of glacial acetic acid may have to be added to 0.1 M KH2P04buffer, pH 7.0 50 ml
the KI solution to induce or improve staining. An 0.1 M MgC12 1 ml
alternative CAT stain was described by Harris 10 mg/ml L-leucine-
and Hopkinson (1976) and Aebersold et al. naphthylamide HCl 0.1 ml
(1987). This enzyme cannot be resolved on high
pH gels. Then add:
fast garnet GBC (dissolved in a
small quantity of water) 0.03 g
Continue incubation.
Dimer The following stain may also yield adeny- This stain was modified from those of Brewer
late kinase (AK) gene products. A control slice (1970), Shaw and Prasad (1970) and Ayala et al.
from the same gel must be stained specifically for (1972). Some of these staining methods involve a
AK to ascertain, by a process of elimination, preincubation step in a boric acid solution which
which zones of activity are CK. This stain may be may not be necessary.
prepared as an agar overlay.
0.2 M Tris-HC1, pH 7.0 50 ml
0.1 M MgC12 1ml
adenosine 5'-diphosphate 0.03 g
glucose 0.05 g
hexokinase 200 u Monomer or Dimer This enzyme was known for-
merly as diaphorase (DIA) and lipoamide dehy-
phosphocreatine 0.05 g
drogenase (EC; see Muramatsu et al., 1978).
10 mg/ml NAD 1ml 0.2 M Tris-HC1, pH 8.0 50 ml
5 mg/ml NBT 1ml 2 mg/ml2,6-dichlorophenol-
5 mg/ml PMS 1 ml indophenol 1 ml
NADH 0.01 g
This stain was described by Buth and Murphy 5 mg/ml MTT 1 ml
(1980) as modifed from Shaw and Prasad (1970).
A more sensitive, but more expensive, fluorescent Zones of enzyme activity will appear pink/pur-
stain was described by Harris and Hopkinson ple against the blue background of the gel. The
(1976). blue DCIP color will clear overnight if the devel-
oped gel is kept refrigerated (dark) yielding a
white gel with purple isozymes. This stain was
GyLosol Aminspcptidase (CAP) $ modified from those of Kaplan and Beutler
QEC3,4,111.1) (1967) and Brewer (1970).Harris and Hopkinson
(1976) used this stain to resolve NADH di-
Monomer This enzyme was known formerly as
aphorase (a synonym of cytochrome-b5 reduc-
leucine aminopeptidase (LAP); the current
tase; EC Aebersold et al. (1987) noted
IUBNC (1984) name and EC number may be
that this stain may also resolve xanthine oxidase
changed as more is learned about peptidases.
(XO) gene products as well as those of a variety
Richardson et al. (1986) refer to this enzyme as
of other enzymes.
Pep-E (see Peptidase). A conservative approach
102 Chapter 4 / Murphy, Sites, Buth & Haufler

Enolasc (ENO) $$$ D.E. Soltis et al., 1983; Shaklee and Keenan, 1986;
(EC4.2.1-11) Aebersold et al., 1987):

Dimer Prepare this stain as an agar overlay. acetic acid, pH 5.5 10 ml

4-methylumbelliferyl acetate 0.01 g
0.2 M Tris-HC1, pH 8.0 50 ml
0.1 M MgC12 5 ml Incubate for approximately 10-30 min at 37OC and
adenosine 5'-diphosphate 0.05 g then view under UV light (long wavelength).
D(-)3-phosphoglycericacid 0.05 g Zones of activity will appear as bright areas. Ad-
just the pH of the acetic acid with sodium acetate.
NADH 0.04 g
pyruvate kinase 20 U
L-lacticdehydrogenase 125 U Formaldehyde Dehydrogenase (FDH) $
phosphoglycerate mutase 125 U (EC 1,2,Z,lb
Monitor the development of expression under UV Dimer
light (long wavelength). Enzyme activity is indi-
cated by zones of defluorescence. This stain was 0.2 M Tris-HC1, pH 8.0 50 ml
modified from Harris and Hopkinson (1976).Two 37% formaldehyde (reagent grade) 3 drops
alternative agar-overlay, fluorescent stains have glutathione, reduced 0.05 g
been described for EN0 that call for slightly dif- 10 mg/ml NAD 3 ml
ferent sets of components that may be less expen- 5 mg/ml NBT 1 ml
sive (Siciliano and Shaw, 1976; Shaklee and 5 mg/ml PMS 1 ml
Keenan, 1986; Aebersold et al., 1987). Prepare the stain and incubate the gel slice at 37OC
in the dark in a fume hood. This stain was modi- ,

fied from an unpublished formula of G.P.

Manchenko (personal communication). See also
Manchenko (1988).
Monomer or Dimer The following general esterase
stain can resolve a number of gene products with
broad substrate specificities; these enzymes might
be identified specifically using other methods.
The products resolved non-specifically might be Dimer or Tetramer This enzyme was known for-
considered as generic carboxylic ester hydrolases merly as hexosediphosphatase (HDP) or fructose-
(EC 3.1.1.-; IUBNC, 1984). 1,6-diphosphatase.
0.2 M Tris-HC1, pH 7.0 50 ml 0.2 M Tris-HC1, pH 8.0 50 ml
a-naphthyl acetate solution 3 ml MgS04*7H20 0.25 g
fast blue BB salt 0.05 g D-fructose-1,6-diphosphate 0.02 g
Incubate at ambient temperature; dark not re- glucose-6-phosphate isomerase 50 U
quired. To prepare the stock substrate solution G6PDH 40 NAD U
(1% solution in 50%acetone), dissolve the a-naph- 2-mercaptoethanol(1 drop in
thy1 acetate in the acetone, then add the water. 10 ml H20) 1 drop
Clearer resolution of some esterase products can NADP 0.02 g
be achieved by using Pnaphthyl acetate as the 5 mg/ml NBT 1 ml
substrate. This stain was modified from Brewer 5 mg/ml PMS 1 ml
(1970).A fluorescent stain for dimeric esterase-D
(EC 3.1.1.-; $) has been described by several in- Prepare stain and incubate the gel slice at 37OC in
vestigators (e.g., Harris and Hopkinson, 1976; the dark in a fume hood. This stain is modified
Proteins: Isozyme Electrophoresis 103

from Shaw and Prasad (1970).This enzyme seems

particularly sensitive to freezing and thawing of
tissue samples.

0.1 M phosphate-citrate buffer, pH 4.0 5 ml

4-methylumbelliferyl-a-D-galactoside 0.01 g
Incubate for approximately 30 min and then view
under UV light (long wavelength). Zones of activ-
Tetramer This enzyme was known formerly as al- ity will appear as bright areas. To enhance fluo-
dolase (ALD) and is currently abbreviated as rescence, spray the gel slice with a concentrated
FBALD by some investigators. ammonium hydroxide solution. This stain was
0.2 M Tris-HC1, pH 8.0 50 ml modified from Harris and Hopkinson (1976).
dehydrogenase 200 U
D-fructose-l,6-diphosphate 0.08 g
10 mg/ml NAD 2 ml
5 mg/ml NBT 1 ml Monomer
5 mg/ml PMS lml 0.1 M phosphate-citrate buffer, pH 4.0 5 ml
This stain was modified from Ayala et al. (1972). 4-methylumbelliferyl-mgalactoside 0.01 g
Aebersold et al. (1987) recommended the addition Incubate at 37OC for approximately 30 min and
of 0.01 g arsenic acid to an FBA stain of 50 ml. then view under UV light (long wavelength).
Zones of activity will appear as bright areas. To
enhance fluorescence, spray the gel slice with a
concentrated ammonium hydroxide solution.
This stain was modified from Harris and Hopkin-
Tetramer This enzyme was known formerly as fu- son (1976).
marase (FUM).The following stain may also yield
malate dehydrogenase (MDH) gene products. A
control slice from the same gel must be stained General Piroteira~s(GP) $
specifically for MDH to ascertain, by a process of [non-specific]
elimination, which zones of activity are FUMH. Various quaternary structures Creatine kinase gene
0.2 M Tris-HC1, pH 8.0 products (Ck-A)and other proteins that predomi-
fumaric acid nate will also stain via this procedure.
malic dehydrogenase Prepare stock solution:
10 mg/ml NAD
5 mg/ml NBT naphthol blue black (amido black) 1g
stain-fixing solution (1:5:5 glacial
5 mg/ml PMS
acetic acid:methanol:water) 500 ml
This stain was modified from Brewer (1970). The
Filter stain to remove undissolved dye.
FUMH stain described by Siciliano and Shaw
(1976) called for the use of the disodium salt of fu- Stain gel slices in 50 ml of stock solution for 20
maric acid and MDH in greater concentration. Ae- min at 20°C. Wash slices in fixing solution several
bersold et al. (1987) recommended the addition of times until background is pale. The stain may be
0.01 g pyruvic acid to a FUMH stain of 50 ml to reused. This stain was modified from Selander et
suppress LDH. al. (1971).
104 Chapter 4 1Murphy, Sites, Buth & Haufler

Glucose Dekydsogenase $GCDH)$ D-fructose-6-phosphate 0.04 g

BEG 1,2,1.138 ) G6PDH 40 NAD U
10 mg/ml NAD 2 ml
Dimer? This enzyme was known formerly as hex-
5 mg/ml NBT 1 ml
ose-6-phosphate dehydrogenase (H6PDH). The
following stain may also yield LDH. Either a con- 5 mg/ml PMS 1 ml
trol slice from the same gel or the addition of 0.05 This stain was described by Buth and Murphy
g pyruvic acid may be necessary. (1980) as modified from DeLorenzo and Ruddle
0.05 M potassium phosphate buffer, (1969).
pH 7.0 50 ml
glucose 9g
10 mg/ml NAD 2 ml
5 mg/ml NBT 1 ml
5 mg/ml PMS 1 ml Tetramer

This stain was modified by Berg and Buth (1984) 0.1 M phosphate-citrate buffer, pH 4.0 5 ml
from that described by Harris and Hopkinson 4-methylumbelliferyl-a-D-glucoside 0.01 g
Monitor the development of expression under UV
light (long wavelength). Zones of activity will ap-
Dehy drogcnase
Glucose-6-P%r;osphaBe pear as bright areas. To enhance fluorescence,
(c6rDssri-a) $st$ spray the gel slice with a concentrated solution of
ammonium hydroxide. This stain was modified
from Harris and Hopkinson (1976). Aebersold et
Dimer? NADP (0.02 g m 400 ml) should be added al. (1987) recommended a stain buffer pH of 8.0.
to the gel before electrophoresis.
0.2 M Tris-HC1, pH 8.0 50 ml
0.1 M MgC12 3 ml
D-glucose-6-phosphate 0.3 g
NADP 0.03 g Subunit strucfure uncertain
5 mg/ml NBT 1 ml 0.1 M phosphate-citrate buffer, pH 4.0 5 ml
5 mg /ml PMS 1ml 4-methylumbelliferyl-bglucoside 0.01 g
This stain was modified from Brewer (1970). At Incubate for approximately 30 min and then
least for amphibians, the quantities of NADP and view under UV light (long wavelength). Zones
D-glucose-6-phosphatemay be reduced by 60% or of activity will appear as bright areas. To en-
more. hance fluorescence, spray the gel slice with a
concentrated ammonium hydroxide solution.
This stain was modified from Harris and Hop-
GIaaccl~e-6-8~1-aospl1ate kinson (1976).
(GFl) $$$
dEC 5,3.%,9)
Dimer This enzyme was known formerly as phos-
phohexose isomerase (PHI) or phosphoglucoiso-
merase (PGI). This stain may be prepared as an Tetramer Some investigators abbreviate this en-
agar overlay. zyme as GUS.
0.2 M Tris-HC1, pH 7.0 50 ml 0.1 M phosphate-citrate buffer, pH 4.0 5 rnl
0.1 M MgC12 5 ml 4-methylumbelliferyl-rnglucuronide 0.01 g
Proteins: Isozyme Electrophoresis 105

Incubate for approximately 30 min and then view 1.0 M L-glutamicacid

under UV light (long wavelength). Zones of activ- 10 mg/ml NAD
ity will appear as bright areas. To enhance fluo- 5 mg/ml NBT
rescence, spray the gel slice with a concentrated 5 mg/ml PMS
ammonium hydroxide solution. This stain was
modified from Harris and Hopkinson (1976).A This stain was modified from Shaw and Prasad
more expensive, non-fluorescent stain was de- (1970). Brewer (1970) recommended a staining
scribed by Aebersold et al. (1987). buffer of pH 9.0. Aebersold et al. (1987) recom-
mended the addition of 0.014 g adenosine 5'-
diphosphate and 0.001 g pyridoxal5-phosphate
Glutamate-Ammonia Ligase (GLAL) $$ to a GTDH stain of 50 ml. A stain buffer of 0.2 M
4EC 6,3,1,2) Tris/HCl pH 7.0 may give equally good results.
Subunit structure uncertain This enzyme was
known formerly as glutamine synthetase (GS).
Preparation of a substrate solution and a visual-
ization solution is required. For the substrate so-
lution, dissolve 0.10 g MgC12*6H20in 2.0 ml H20,
then add: Subunit structure uncertain
0.2 M Tris-HC1 buffer, pH 8.0 3 rnl 0.1 M potassium phosphate buffer,
L-glutamicacid 0.20 g pH 7.0 35 ml
adenosine 5'-triphosphate 0.08 g 1.0 M L-glutamicacid 15 ml
NH40H (concentrated) 0.2 ml NADP 0.02 g
5 mg/ml NBT 1 ml
Raise the pH to 9.3 with 1.0 N NaOH. Incubate
5 mg/ml PMS 1 ml
the gel slice in the substrate solution at 37OC for at
least 1 hr, then remove the substrate solution from Counterstain for GTDH. This stain was modified
the gel slice but do not rinse. Cover the gel slice from Shaw and Prasad (1970). Brewer (1970) rec-
with a 50% acetone solution and incubate it at am- ommended a staining buffer of pH 9.0. Aebersold
bient temperature for 15 min. Flush the gel slice et al. (1987) recommended the addition of 14 mg
with water and add the following visualization adenosine 5'-diphosphate and 1 mg pyridoxal5-
solution: phosphate to a GTDHP stain of 50 ml volume.
L-ascorbicacid 0.8 g
ammonium molybdate solution
(see below) 6 ml
Incubate at 37OC. The ammonium molybdate so-
lution can be prepared as a stock (2.5 g molybdic
Dimer Prepare as an agar overlay (0.17 g agar in
acid ammonium tetrahydrate, 8 ml concentrated
10 ml water, mix the stain components in the 13
H2SO4,92 ml water). This stain was described by
ml buffer):
Morizot et al. (1983).

0.2 M Tris-HC1, pH 8.0 13 ml

2 mg/ml2,6-dichlorophenol-
indophenol 1 ml
glutathione, oxidized 0.02 g
flavine adenine dinucleotide 0.002 g
0.1 M potassium phosphate buffer, NADH 0.01 g
pH 7.0 35 ml
5 mg/ml MTT 1 ml
106 Chapter 4 / Murphy, Sites, Buth t3 Haufler

This stain was modified from Aebersold et al. gene products. A control slice from the same gel
(1987).Alternative stains are discussed by Brewer may be necessary.
(1970). P.T. Chippindale (personal cokmunica-
0.2 M Tris-HC1, pH 8.0
tion) obtains better results by applying the stain
without agar and in only 13 ml of Tris/HCl. Di-
hydrolipoamide dehydrogenase (DDH; EC pyruvic acid may also be resolved as a second, rela- pyrazole
tively slower system (see Harris and Hopkinson, 10 mg/ml NAD
1976) because it appears if glutathione is omitted 5 mg/ml NBT
from the stain. In addition, because Aebersold et 5 mg/ml PMS
al. (1987) noted that the DDH stain may also re- This stain was modified from Siciliano and Shaw
solve xanthine oxidase (XO) gene products as well (1976).
as those of a variety of other enzymes, this may
also be true for GR. FAD may not be necessary if
NADPH is used instead of NADH. However, the Glycerol-3-Phosp11aPe Dehydrogenase
use of NADPH may result in the resolution of (G3PDHB $$
NADPH dehydrogenase (EC (EC 1..3 .I .$)
Dirner This enzyme was known formerly as
a-glycerophosphate dehydrogenase (aGPD or
0.2 M Tris-HC1, pH 8.0 50 ml
Tetrarner Prepare the substrate solution: DL-glycerophosphate,pH 8.0 lg
0.1 M MgC12 1ml
0.2 M Tris-HC1, pH 7.0 10 ml
aldolase 100 U 10 mg/ml NAD 1 ml
D-fructose-1,6-diphosphate 0.25 g 5 mg/ml NBT 1 ml
5 mg/ml PMS 1 ml
Incubate the substrate solution at 37'C for 30
min, then add the following: This stain may require a higher pH of stain buffer
(pH 9.5), 2 ml of NAD, and incubation for 1 hr be-
0.2 M Tris-HC1, pH 7.0 fore adding PMS. This stain was modified from
arsenic acid Shaw and Prasad (1970).
10 mg/ml NAD
5 mg/ml NBT
5 mg/ml PMS
Guanine Deami.na.se(GDA) $$
4EC 3.5,4.3)
This stain was modified from those of Ayala et al.
(1972) and Siciliano and Shaw (1976). In order to Dirner This stain may be applied as an agar over-
minimize L-lactate dehydrogenase (LDH) stain- lay (use 0.07 g agar in the 5 ml water and mix the
ing, 0.1 g of pyruvic acid may be added to the stain components in the 5 ml buffer). Otherwise
staining solution (Harris and Hopkinson, 1976). simply mix the following:
0.2 M Tris-HC1, pH 8.0
Glycerate Dehydrogenase Hz0
(GLYDH) $$$$
xanthine oxidase
(EC 1.1,1.29) 5 mg/ml MTT
Subunit structure uncertain The following stain 5 mg/ml PMS
may also yield L-lactate dehydrogenase (LDH)
Proteins: Isozyme Electrophoresis 107

If the enzyme activity is low, the amount of XO 10 mg/ml NAD 2 ml

may have to be increased. However, Harris and 5 mg/ml NBT 1 ml
Hopkinson (1976) recommended that less XO be 5 mg/ml PMS lml
used (0.1 U). This stain was modified from
Richardson (1983). Incubate the gel slice in the dark at ambient tem-
perature or at 37OC. This stain was modified from
Shaw and Prasad (1970). D.E. Soltis et al. (1983)
Coranylate Kinase (GUK) $$$ and Werth (1985) suggested that 0.02 g tetra-
(EC sodium EDTA be added to an HK stain of 50 ml.
Monomer This stain may be prepared as an agar
overlay (0.07 g agar in 5 ml of buffer and mix the D-2-Hydroxy- Acid Deh ydrogenase
stain components in the remaining 8 ml of buffer). (HADHB $
0.2 M Tris-HC1, pH 8.0 (EC 1,1,99.6)
0.1 M MgC12 Dimer This enzyme has multiple substrate affini-
adenosine 5'-triphosphate ties, and may be confused with 3-hydroxybu-
guanosine 5'-monophosphate tyrate dehydrogenase (HBDH). Both LDH and
phospho(eno1)pyruvate ADH gene products may appear with prolonged
NADH staining. Either a control slice from the same gel
potassium chloride or end-product suppression staining may be nec-
calcium chloride essary.
pyruvate kinase Dissolve 2.0 g D-gluconic acid lactone in 25 ml
lactic dehydrogenase water. Adjust the p H to 12.5 with sodium
Monitor the development of expression under UV hydroxide pellets ( ~ 0 . 5g). Incubate this solu-
light (long wavelength). Enzyme activity is indi- tion at ambient temperature for 30 min with
cated by zones of defluorescence. This stain was occasional stirring. Then readjust the pH to just
modified from Harris and Hopkinson (1976). See below 8.0 by adding, dropwise, 12 N HCl. Add
also Morizot and Siciliano (1982). 25 ml of 0.2 M Tris-HC1, pH 8.0 (at this point,
the pH of the substrate solution should be 8.0
but a minor adjustment with 4 N HCl may be
necessary). Add the following to the substrate
solution and apply to the gel slice:
Monomer The following stain may also yield glu- pyrazole 0.05 g
cose dehydrogenase (GCDH) gene products. Ei- pymvic acid 0.05 g
ther a control slice from the same gel must be 10 mg/ml NAD Iml
stained specifically for GCDH to ascertain, by a 5 mg/ml NBT lml
process of elimination, which zones of activity are 5 mg/ml PMS 1 ml
HK, or 0.05 g of gluconic acid must be added to
the stain to suppress GCDH. HK and GCDH of- This stain was described by Buth (1980) as modi-
ten have very different tissue-specific expression fied from Tobler and Grell(1978).
so the choice of tissue may make continued con-
trol testing unnecessary.
0.2 M Tris-HC1, pH 8.0 50 ml
0.1 M MgC12 1ml
Tetramer This enzyme was known formerly as
adenosine 5'-triphosphate 0.25 g glycolate oxidase (GOX) EC Prepare as an
D(+)-glucose 5g agar overlay.
108 Chapter 4 /Murphy, Sites, Buth & Haufler

0.2 M Tris-HC1, pH 8.0 0.1 M MgC12 2 ml

glycolic acid NaCl 0.30 g
5 mg/ml MTT DL-hydroxybutyricacid 0.63 g
5 mg/ml PMS 10 mg/ml NAD 3 ml
This stain was modified from R.L. Garthwaite 5 mg/ml NBT 1 ml
(personal communication). 5 mg/ml PMS 1 ml
This stain was modified from Shaw and Prasad
Hydrolase (1970).
(EC 3.3..2.6) L-IditolDehydrogenase (IBBH) $
Dimer This enzyme was known formerly as gly- (EC 1,3..l.I4B
oxalase I1 (GLO-11).This stain should be prepared Tetramer This enzyme was known formerly as sor-
as an agar overlay (0.17 g agar in 10 rnl water; mix bit01 dehydrogenase (SDH or SORD). LDH gene
the stain components in the 15 ml buffer): products may appear with prolonged staining.
0.2 M Tris-HC1, pH 8.0 Either a control slice from the same gel or end-
methylglyoxal product suppression staining may be necessary.
glutathione, reduced 0.2 M Tris-HC1, pH 8.0
glyoxalase I 2.0 M D-sorbitol
10 mg/ml NAD
Incubate at 37OC for at least 30 min, then add:
5 mg/ml NBT
1.0 M zinc chloride 5 mg/ml PMS
pyruvic acid
This stain was modified from Lin et al. (1969).En-
L-lacticdehydrogenase zyme products often migrate cathodally.
10 mg/ml NAD
5 mg/ml MTT
5 mg/ml PMS
Incubate the gel slice at 37OC in the dark. This
stain was modified from Aebersold et al. (1987). Dimer The following stain is for NADP-depen-
dent isocitrate dehydrogenase. Mitochondria1 and
supernatant/cytosolic forms are known (Harris
3-~ydroxg;baatyrateD~hydragenase and Hopkinson, 1976).
0.2 M Tris-HC1, pH 8.0 50 ml
0.1 M MgC12 3 ml
Dimer This enzyme may be confused with 2- DL-isocitricacid 0.08 g
hydroxy-acid dehydrogenase (HADH) due to the NADP 0.01 g
multiple substrate affinities of the latter. A control 5 mg/ml NBT 1 ml
slice from the same gel should be stained for 5 mg/ml PMS 1 ml
HADH. ~lectromor~hs in common between slices
stained for HBDH and HADH are probably This stain was modified from those of N.S. Hen-
HADH. ADH products may appear with pro- derson (1965), Brewer (1970), Shaw and Prasad
longed staining. (1970), and Ayala et al. (1972). Note that Brewer
(1970) erred in listing manganese chloride
0.5 M potassium phosphate buffer, (MnC12)instead of magnesium chloride in this
pH 7.0 25 ml stain. For amphibians, the Tris-HC1buffer should
Hz0 20 ml be adjusted to pH 7.0.
Proteins: Isozyme Electrophoresis 109

5 mg/ml NBT 1 ml
5 mg/ml PMS 1 ml
Tetramer This stain was modified from Brewer (1970).
0.2 M Tris-HC1, pH 8.0 50 ml
1.0 M lithium lactate, pH 8.0
(see below) 8 ml
10 mg/ml NAD lml
5 mg/ml NBT lml
5 mg/ml PMS lml
Tetramer The following stain is for NADP-depen-
dent malate dehydrogenase. The convention for
The stock substrate solution may be prepared us- name abbreviation of this kind of enzyme (+P to
ing either DL-lacticacid or lactic acid solution; the MDH) follows Aebersold et al. (1987). This en-
pH should be adjusted to 8.0 with the addition of zyme was known formerly as malic enzyme (ME).
LiOH. This stain was modified from Shaw and Mitochondrial and supernatant/cytosolic forms
Prasad (1970). are known (Harris and Hopkinson, 1976). NADP
(0.02 g in 400 ml) should be added to the gel be-
fore electrophoresis.
0.2 M Tris-HC1, pH 8.0 50 ml
0.1 M MgC12 1 ml
Dimer This enzyme was known formerly as gly- 2.0 M DL-malicacid, pH 8.0 5 ml
oxalase I (GLO). NADP (see note below) 0.02 g
0.2 M potassium phosphate buffer, 5 mg/ml NBT 1 ml
pH 6.8 50 ml 5 mg/ml PMS 1 ml
methylglyoxal(40%solution) 0.9 ml
This stain was modified from those of Ayala et al.
glutathione, reduced 0.25 g
(1972) and Cross et al. (1979).It is important that
5 mg/ml MTT 1 ml NADP be used in solid form in this stain. There is
Incubate the gel slice in this solution at 37OC for often sufficient breakdown of NADP to NAD in
40 min and then add: liquid stocks in prolonged storage that NAD-de-
pendent MDH activity will be resolved in addition
2 mg/ml2,6-dichlorophenol- to MDW. If there is any doubt as to the identity of
indophenol 1 ml MDHP, a control slice from the same gel should be
Areas of activity will be seen as white zones on a stained specifically for MDH to ascertain, by a
blue background. This stain was modified from process of elimination, which zones of activity are
Harris and Hopkinson (1976). MDHP. Aebersold et al. (1987) recommended
adding 0.02 g oxaloacetic acid to an MDHP stain
of 50 ml. Use caution when preparing the DL-malic
acid substrate as the solution becomes extremely
hot while adjusting the pH with NaOH.

Dimer The following stain is for NAD-depen-

dent malate dehydrogenase. Mitochondria1and Mannsse-6-Phosph,7te Isemerase
supernatant/cytosolic forms are known (Harris (1VPsr)$$$$
and Hopkinson, 1976). (EC 5-3.1-8)
0.2 M Tris-HC1, pH 8.0 Monomer This stain may be prepared as an agar
2.0 M DL-malicacid overlay.
10 mg/ml NAD
110 Chapter 4 / Murphy, Sites, Buth & Haufler

0.2 M Tris-HC1, pH 8.0 50 ml keep the gel slice moist (Harris and Hopkinson,
0.1 M MgC12 1 ml 1976; Aebersold et al., 1987). After incubation,
D-mannose-6-phosphate 0.05 g remove the substrate solution from the slice
glucose-6-phosphate isomerase 50 U but do not rinse. Add the following visualiza-
G6PDH 40 NAD U tion solution:
10 mg/ml NAD 2 ml L-ascorbic acid 0.5 g
5 mg/ml MTT 1 ml ammonium molybdate solution
5 mg/ml PMS 1ml (see below) 2 ml
Products of LDH may appear as faint bands fol- Incubate at 37OC in a fume hood in the dark. The
lowing staining. LDH activity can be suppressed ammonium molybdate solution can be prepared
by adding 0.05 g of pyruvic acid. This stain was as a stock (2.5 g ammonium molybdate, 8 ml con-
described by Buth and Murphy (1980) as modi- centrated H2SO4,92 ml H20).This stain was mod-
fied from E. Nichols et al. (1973). ified from Aebersold et al. (1987).

a-Manncasidase (rkMAN) $$$$

(EC 3-2.1-24)
Monomer or dimer Dimer
0.1 M phosphate-citrate buffer, pH 4.0 5 ml 0.2 M Tris-HC1, pH 8.0
4-methylumbelliferyl-a- 1-octanol
D-mannopyranoside 0.01 g 95% ethanol
Incubate for 30 rnin and then view under UV light 10 mg/ml NAD
(long wavelength). Zones of activity will appear 5 mg/ml NBT
as bright areas. To enhance fluorescence, spray the 5 mg/ml PMS
gel slice with a concentrated ammonium hydrox- This stain, modified from those of Ayala et al.
ide solution. This stain was modified from Harris (1972) and Shaklee and Keenan (1986), includes
and Hopkinson (1976). ethanol, which may also serve as a substrate for
alcohol dehydrogenase (ADH). A control slice
from the same gel should be stained for ADH us-
ing only ethanol as substrate to ascertain which
zones of activity are ODH.

Dimer This enzyme was known formerly as ino-

sine triphosphatase (ITP). This stain requires the
u-Oelopine Dehydrogrrnase (OPDH) $$
preparation of a substrate solution and a visual- (EC
ization solution. Monomer Prepare this stain as an agar overlay
Substrate solution: (0.17 g agar in 15 ml buffer, the stain components
in the remaining 10 rnl buffer):
0.2 M Tris-HC1, pH 8.0 15 ml
MgC12-6H20 0.1 g 0.2 M Tris-HC1, pH 8.0 25 ml
inosine triphosphate 0.03 g 0.1 M MgC12 1 ml
2-mercaptoethanol 120 pl D-octopine 0.01 g
10 mg;/ml
NAD 1 ml
Mix these components and pour on the gel slice 5 mg/ml NBT
in a fume hood. Incubate at 37OC for at least one mg/ml pMS
hour. A filter paper overlay may be desired to
Proteins: Isozyrne Electrophoresis 111

This stain was modified from Shaklee and Keenan Snake venom is used in this stain as a source of L-
(1986). This enzyme is known only from inverte- amino acid oxidase. The substitution of a less pu-
brates. rified but adequate source of this enzyme (via
snake venom) was advantageous financially at
one time but may no longer be so. Several stain
Pep tidase (PEE3) formulas list specifically the venom of the eastern
(EC 3.4.-,-) diamondback rattlesnake (Crotalus adamanteus) for
Subunit structure variable The terms dipeptidase use in peptidase stains (e.g., Siciliano and Shaw,
(EC and tripeptide aminopeptidase 1976).We have tried the less expensive venom of
(EC are recommended over the more a closely related rattlesnake, the western dia-
generic term peptidase (IUBNC, 1984).However, mondback (C. atrox, recommended herein), and
the multiple substrate affinities of these enzymes found that it yielded equivalent results. These
and problematic assignment of their homology stains may disappear quickly and thus should be
makes the exact assignment of EC numbers diffi- scored and photographed promptly. For rapidly
cult. Exceptions are those of proline dipeptidase developing peptidases (e.g., Pep-A), it may be ad-
(Pep-D; EC and perhaps cytosol vantageous to incubate these gels at room tem-
aminopeptidase (Pep-E; EC 3.4.11.I). Recom- perature to slow staining.
mended substrates for the resolution of products
of seven peptidase loci described from verte- Feroxidase (PER) $
brates follow Frick (1981,1983, personal commu-
nication), Richardson et al. (1986), and/or Mat-
(EC 1.1.3.,1.7)
son (1989). The tissue distribution of these gene Subunit structure uncertain
products is often restricted (e.g. Frick, 1983; Mat-
3-amino-9-ethyl carbazole
son, 1989). Pep-B frequently appears upon stain-
ing for Pep-F, as does Pep-C on Pep-A making
counterstaining necessary. Then add:
0.05 M sodium acetate buffer, pH 5.0 5 ml
Pep-A: glycyl-L-leucine[dimerl $$$
0.1 M calcium chloride 1 ml
Pep-B: L-leucylglycylglycine[monomer or
dimerl $$$ 3% hydrogen peroxide 1 rnl
Pep-C: glycyl-L-leucine or DL-alanyl-DL- Incubate the gel slice in a refrigerator, usually for
methionine [monomer]$$$ 30-60 min. This stain was modified from Shaw
Pep-D: L-phenylalanyl-L-proline[dimerl $$$ and Prasad (1970) by D.E. Soltis et al. (1983). See
Pep-E: see cytosol aminopeptidase [monomer] Brewer (1970) and Siliciano and Shaw (1976) for
Pep-F: L-leucyl-L-leucyl-L-leucine[subunit additional PER stains.
structure unknown] $$$$
Pep-S: glycyl-L-leucine[tetramer?] $$$

The following general stain for peptidases is mod-

ified from Merritt et al. (1978) and is best used as Dimer This stain may be prepared as an agar
an agar overlay: overlay (0.14 g agar in 10 ml of water and stain
components in the remaining 10 ml of buffer).
0.2 M Tris-HC1, pH 8.0 50 ml
di/tripeptide (see above) 0.04 g 0.2 M Tris-HC1, pH 8.0 10 ml
snake venom (from Crotalus atrox) 0.01 g Hz0 10 ml
peroxidase 0.02 g D-fructose-6-phosphate 0.012 g
o-dianisidine dihydrochloride 0.01 g adenosine 5'-triphosphate 0.012 g
MgC12*6H20 0.04 g
112 Chapter 4 / Murphy, Sites, Buth & Haufler

arsenic acid (Na salt) 0.20 g

2-mercaptoethanol 20 pl
aldolase 36 U
triosphosphate isomerase 500 U
a-glycerophosphatedehydrogenase 40 U Dimer This enzyme was known formerly as 6-
phosphogluconate dehydrogenase (6PGD or
10 mg/ml NAD 1 ml
6PGDH). B.J. Turner (1974) identified a second
Prepare stain and incubate the gel slice at 37OC in gene product that sometimes appears with this
a fume hood. Incubate for 30 min and then view stain as glucose-6-phosphate dehydrogenase.
under UV light (long wavelength).Zones of activ- Limit the following staining solution to the imme-
ity will appear as bright areas. To enhance fluo- diate area of anticipated activity. NADP (0.02 g in
rescence, spray the gel slice with a concentrated 400 ml) should be added to the gel before elec-
ammonium hydroxide solution. This stain was trophoresis.
modified from Harris and Hopkinson (1976),who
recommend the addition of 2-mercaptoethanol (fi- 0.2 M Tris-HC1, pH 8.0 5 ml
nal concentration of 10 mM) and ATP (final con- 0.1 M MgC12 5 ml
centration of 0.2 mM) to gel before degassing, but 6-phosphogluconic acid 0.01 g
this may not be necessary. NADP 0.01 g
5 mg/ml NBT 0.5 ml
5 mg/ml PMS 0.5 ml
"(PGM) $$
(EC This stain was modified from Shaw and Prasad
(1970). Harris and Hopkinson (1976) recom-
Monomer This stain may be prepared as an agar mended an agar overlay for PGDH. The use of
overlay. high amperage may result in sigruficant streaking.
0.2 M Tris-HC1, pH 8.0 50 ml
0.1 M MgC12 5 ml
a-~glucose-1-phosphate 0.1 g
10 mg/ml NAD 2 ml Monomer This stain should be prepared as an
5 mg/ml NBT 1 ml agar overlay (0.14 g agar in 10 ml buffer and stain
components in the remaining 10 ml buffer).
5 mg/ml
- PMS 1 ml
0.2 M Tris-HC1, pH 8.0 20 ml
This stain was described by Buth and Murphy
0.1 M MgC12 1 ml
(1980) as modified from N. Spencer et al. (1964).
The reaction requires glucose-1,6-diphosphate in D(-)3-phosphoglycericacid 0.03 g
addition to glucose-1-phosphate listed above. adenosine 5'-triphosphate 0.05 g
However, the former biochemical is expensive if NADH 0.02 g
purchased in purified form and only a trace is glyceraldehyde-3-phosphate
necessary. The Sigma G-7000 product is said to dehydrogenase 20 U
carry a trace of G-1,6-P (0.01-0.2%)which is usu- a-glycerophosphate dehydrogenase 5U
ally enough to stain for PGM but may not be so in Monitor the development of expression under UV
some cases. D.E. Soltis et al. (1983) and Werth light (long wavelength). Staining may occur
(1985) recommended the Sigma G-1259 product rapidly; check after 5-10 min. Enzyme activity is
which, while more expensive than G-7000, has a indicated by zones of defluorescence. This stain
sufficient amount of G-1,6-P (1%)as a contami- was modified from those of Beutler (1969) and
nant to guarantee PGM activity. Harris and Hopkinson (1976). Aebersold et al.
(1987) recommended using 10 times this amount
Proteins: Isozyme Electrophoresis 113

of magnesium chloride and adding 300 U of 0.05 M potassium phosphate buffer,

triose-phosphate isomerase. We have found the pH 7.0 50 ml
use of a-glycerophosphate dehydrogenase to be inosine 0.01 g
unnecessary in amphibians, reptiles and mam- xanthine oxidase 0.4 U
mals. A stock partial substrate buffer solution may 5 mg/ml MTT 1 ml
be prepared as follows: 5 mg/ml PMS 1 ml
MgCl2*6H20 Incubate the gel slice at 37OC in the dark. This
EDTA stain was modified from those of Harris and Hop-
0.2 M Tris-HC1, pH 8.0 kinson (1976) and Ward et al. (1979).
Store partial substrate solution under refrigera-
tion. Add 3-phosphoglyceric acid, NADH, ATP,
and GAPDH to the partial substrate solution in
the quantities described above, incubate and view
under UV light (long wavelength). Subunit structure uncertain
0.2 M Tris-HC1, pH 8.0
10 mg/ml NAD
5 mg/ml NBT
5 mg/ml PMS
Dimer This stain was modified from Mulley and Latter
0.2 M Tris-HC1, pH 8.0 (1980).
~(+)2-phosphoglyceric acid
adenosine 5'-triphosphate
disodium EDTA
NADH Tetramer The following stain may also yield
3-phosphoglyceric phosphokinase adenylate kinase (AK) gene products. A control
glyceraldehyde-3-phosphate slice from the same gel must be stained specifi-
dehydrogenase cally for AK. This stain may be prepared as an
Monitor the development of expression under agar overlay.
UV light (long wavelength). Enzyme activity is 0.2 M Tris-HC1, pH 8.0 50 ml
indicated by zones of defluorescence. This stain 0.1 M MgC12 6ml
was modified from those of Harris and Hopkin- adenosine 5'-diphosphate 0.03 g
son (1976) and Siciliano and Shaw (1976).An al- D(+)-glucose 0.09 g
ternative stain is suggested by Richardson et al. hexokinase 200 u
(1986). phospho(eno1)pyruvate 0.02 g
10 mg/ml NAD 1 ml
5 mg/ml NBT 1 ml
5 mg/ml PMS 1 ml
Trimer This enzyme was known formerly as nu- This stain was described by Buth and Murphy
cleoside phosphorylase (NP).This stain should be (1980) as modified from Brewer (1970).This stain
prepared as an agar overlay. may also resolve adenylate kinase gene products.
114 Chapter 4 / Murphy, Sites, Buth & Haufler

A fluorescent stain that develops very rapidly was Superoxide Dismegtase (SOD) $
described by Harris and Hopkinson (1976). (EC 1-15.1*1)
Dimer and tetramer This enzyme was known for-
Retinol Dehydrogexxase (R.DH)$$$ merly as indophenol oxidase (IPO) or tetrazolium
(EC 1.1.1,105) oxidase (TO).Mitochondria1and supernatant/cy-
tosolic forms are known (Harris and Hopkinson,
Subunit structure uncertain
1976). The mitochondria1 form is a tetrameric
retinol manganoprotein, whereas the cytosolic form is a
acetone dimeric cuprozinc protein (Healy and Mulcahy,
Then add to:
0.2 M Tris-HC1, pH 9.0
0.1 M phosphate buffer, pH 7.0 50 ml 0.1 M MgC12
10 mg/ml NAD 1 ml
10 mg/ml NAD
5 mg / rnl NBT 1 ml
5 mg/ml NBT
5 mg/ml PMS 1 ml
5 mg/ml MTT
This stain recipe was supplied by R.L. Garthwaite 5 mg/ml PMS
(personal communication).
Incubate the gel slice exposed to light at ambient
temperature or at 37OC. The enzyme appears as
Shikimate Behydrogencase (SKDH) $$$ light bands on a dark background and is fre-
(EC quently resolved on other enzyme systems (e.g.,
G3PDH, PNP, ACOH). This stain is modified from
Subunit structure uncertain A.G. Johnson et al. (1970) and Siciliano and Shaw
0.2 M Tris-HC1, pH 8.0 50 ml (1976).In some cases, resolution may be improved
shikimic acid 0.05 g if this stain is applied as an agar overlay.
NADP 0.01 g
5 mg/ml NBT 1 ml
5 mg/ml PMS 1 ml
This stain was modified from D.E. Soltis et al. Subunit structure uncertain This enzyme was pre-
(1983). viously called rhodanase (RDS).
0.2 M Tris-HC1, pH 8.0
Succinahe Dekydlrogenase (SUDH) $$ potassium cyanide
(EC 1..3.99.3.) sodium thiosulfate
Monomer 5 mg/ml MTT
5 mg/ml PMS
0.1 M phosphate buffer, pH 7.0 50 ml
adenosine 5'-triphosphate 0.04 g This recipe was given to us by R.D. Sage (personal
succinic acid 0.2 g communication).
disodium EDTA 0.2 g
10 mg/ml NAD 3 ml
5 mg/ml NBT 1 ml
5 mg/ml PMS 1 ml
Dimer This stain may be prepared as an agar
This stain was modified from Brewer (1970). overlay (0.12 g agar in 10 ml buffer, stain compo-
nents in remaining 2 rnl buffer).
Proteins: Isozyme Electrophoresis 115

0.2 M Tris-HC1, pH 8.0 12 ml UTP-Clueose-I-Phosphate

dihydroxyacetone phosphate solution Uri dyly Bransf erase QUGUT)$$$$
(see below) lml
(EC 2.7.7,9)
dehydrogenase 800 U Monomer or octamer This enzyme, formerly
arsenic acid 0.1 g known as uridine diphosphoglucose pyrophos-
10 mg/ml NAD 3 ml phorylase (UDP), is commonly resolved as two
5 mg/ml NBT 0.5 ml isozymes in plants. In humans, this enzyme ap-
pears to be octameric, whereas in plants it is
5 mg/ml PMS 0.5 ml
monomeric. UTP is an abbreviation for uridine
This stain was modified from Brewer (1970). The triphospho and should not be used as an enzyme
required amount of glyceraldehyde-3-phosphate system designation. Prepare as an agar overlay
can often be reduced by 50%. Preparation of the (0.07 g agar in 6 ml buffer and mix the stain com-
dihydroxyacetone phosphate (DHAP) solution ponents in remaining 3 ml buffer and water).
follows Morizot and Schmidt (1990). Rinse 5 g
0.2 M Tris-HC1, pH 8.0 1.5 ml
Dowex-50 resin (supplied with DHAP) four times
H20 7.5 ml
in distilled water. Add 0.25 g DHAP and 5 g
washed Dowex-50 resin to 10 ml water; swirl for uridine 5'-diphosphoglucose 0.04 g
30 sec. Filter into graduated cylinder and add wa- pyrophosphate 3 mg
ter to 25 ml. Incubate at 37OC for 4 hr. Adjust pH a-D-glucose-1,6-diphosphate 0.5 mg
to 4.5 with KHC03. Freeze in 1ml aliquots. An al- phosphoglucomutase 75 u
ternative procedure that does not involve the very G6PDH 10 NAD U
expensive DHAP is described by Ayala et al. 10 mg/ml NAD lml
(1972).The latter procedure requires two hours of 5 mg/ml NBT 1ml
substrate preincubation and two pH adjustments 5 mg/ml PMS 1ml
for each stain preparation.
Stains rapidly with good resolution. Excessive py-
rophosphate will suppress enzyme activity. This
Tyrosine Amino transf erase (TAT) $$$ stain was modified from Harris and Hopkinson
(EC 2,6.3 -5) (1977) and Jech and Wheeler (1984).
Subunit structure uncertain The stain should be pre-
pared as an agar overlay (0.14 g agar in 10 ml H20,
mix the stain components in the 15 ml buffer).
0.2 M Tris-HC1, pH 8.0 15 ml Monomer This enzyme was known formerly as
H20 10 ml uridine monophosphate kinase (UMPK). This
L-tyrosineHC1(1%solution in stain should be prepared as an agar overlay (0.14
0.2 N HC1) 1ml g agar in 10 ml buffer and mix the stain compo-
a-ketoglutaric acid 0.01 g nents in remaining 10 rnl buffer).
adenosine 5'-diphosphate 0.01 g
0.2 M Tris-HC1, pH 8.0
pyridoxal5-phosphate 0.01 g MgC12*6H20
L-glutamicdehydrogenase 100 u uridine 5'-monophosphate
10 mg/ml NAD lml adenosine 5'-triphosphate
5 mg/ml NBT 1 ml phospho(eno1)pyruvate
5 mg/ml PMS lml NADH
This stain was modified from Aebersold et al. (1987) potassium sulfate
and D.E. Campton (personal communication). pyruvate kinase
L-lactic dehydrogenase
116 Chapter 4 /Murphy, Sites, Buth & Haufler

Monitor the development of expression under UV 1. 1:5:5 glacial acetic acid:methanol:water

light (long wavelength). Enzyme activity is indi-
cated by zones of defluorescence. This stain was Caution must be exercised when handling fixed
modified from Harris and Hopkinson (1976). gel slices; do not breathe vapors. Do not soak
(completely immerse) gels stained with MTT in
this fixative or significant fading will result. This
fixative is from Selander et al. (1971).
2. 50% ethanol (in water)
Monomer or dimer XO, LDH, ALDH, and A 0 may
be resolved upon staining for XDH (see Maldon- This should not be used for the fixation of AAT
ado, 1992) which has led to speculations about the and PER stains; see D.E. Soltis et al. (1973).
existence of XDH in vertebrates (Richardson et al., 3. 50% glycerol (in water)
1986). Hypoxanthine is quite insoluble; Brewer
(1970) recommended heating the substrate in the This fixative may be preferred for gels stained us-
buffer and then adding the other components ing MTT as a dye to reduce fading. Soak gels for
when the solution has cooled. Richardson et al. several hours before wrapping if the gels are to be
(1986) recommended suspending the hypoxan- saved. This method of fixation may result in the
thine in acetone (0.04 g hypoxanthine per ml of resolution of faint LDH isozymes (see also D.E.
acetone).Another method to increase the amount Soltis et al., 1983; Werth, 1985).
of substrate in solution requires the preparation of
the following:
0.2 M Tris-HC1, pH 8.0 5 ml
hypoxanthine (see note below) 0.2 g ELECTRBPHQBRESIS
Stir this solution for at least 10 min, then add: (Compiled by Donald G. Buth and Robert W.
0.2 M Tris-HC1, pH 8.0
NADH The importance of buffers has reemerged as a crit-
10 mg/ml NAD ical issue for the resolution of enzymes in starch
5 mg/ml NBT gels. However, the search for optimal buffers of-
5 mg/ml PMS ten leads to a trade-off of cost versus quality. The
more buffers needed for optimal resolution of a
Adjust the pH to 8.0, if necessary. Cover the gel large array of enzymes, the higher the cost in both
slice with the stain solution; make sure that any effort and funds. To illustrate both extremes, Har-
undissolved hypoxanthine covers the gel slice. In- ris and Hopkinson (1976) recommended a differ-
cubate the gel slice at 37OC in the dark. Note that ent buffer, or slight modification thereof, for al-
for some species, as much as 1 g of hypoxanthine most every enzyme system listed, whereas
may be required for optimal resolution. For some Siciliano and Shaw (1976) "worked to develop
organisms, xanthine may be a more suitable sub- just a few buffer systems which would give clear
strate. This stain was modified from those of resolution of many proteins" and reported clear
Brewer (1970), Shaw and Prasad (1970),and Mal- resolution of enzymes using only two buffers;
donado (1992). Tris-citrate, pH 7.0, and Tris-borate-EDTA 11, pH
8.0. Many laboratories have geared their opera-
Stain-Fixing Solutions tions toward the latter extreme, which has re-
sulted in less than "clear resolution" for many en-
Many stain fixing solutions have been used. These zymes of many taxa. We encourage the initial
include the following: screening of enzyme systems using several of the
Proteins: Isozyme Electrophoresis 117

buffer systems listed herein followed by compar- is not necessary to use non-denatured ethanol al-
isons using similar buffers, if necessary, for opti- though this may be preferable to keep methyl and
mal resolution of enzymes. Additional buffers are isopropyl alcohol out of the gel.
listed by Brewer (1970), Selander et al. (1971),
Clayton and Tretiak (1972),Harris and Hopkinson
(1976), Steiner and Joslyn (1979), Shaklee and
Tamaru (1981), Conkle et al. (1982), D.E. Soltis et Stock solution:
al. (1983), Cheliak and Pitel (1984), Werth (1985), (0.04 M) citric acid monohydrate 8.4 g/liter
Micales et al. (19861, Selander et al. (1986), Shak-
lee and Keenan (1986),Aebersold et al. (1987),and Adjust to desired pH by adding =lo-15 ml/liter
Morizot and Schmidt (1990). Several of those N-(3-aminopropy1)-morpholine
listed for use in cellulose acetate electrophoresis Electrode: Undiluted stock solution
by Richardson et al. (1986:153-154) may be ap-
plicable to starch gel work. The reader must re- Gel: 1:19 dilution of stock solution
main aware that buffer formulas are usually de- These gels are hazardous and should be handled
rived empirically and additional modification only with protective gloves. This buffer was de-
should be encouraged. Sambrook et al. (1989) pre- scribed by Clayton and Tretiak (1972). Werth
sented a useful appendix for the preparation of (1985),Shaklee and Keenan (1986), and Aebersold
phosphate buffers. et al. (1987) recommended its use at pH 6.1, 6.0,
Our buffer accounts include a descriptive and 7.0, respectively. Its range of use may be pH
name of the system, molarities of components in 6.0-8.0 (D.E. Campton, personal communication).
solution, exact gram measures of components in Aebersold et al. (1987) suggested the inclusion of
one liter equivalents, formulas for stock solutions 0.01 M EDTA in the stock solution.
as well as dilutions for electrode chambers and
the gels, and references. We have resisted listing
the electrical potential for each of these buffer
systems, although many other compilations of Stock solution:
buffer formulas provide such information. (0.04 M) citric acid monohydrate 8.4 g/liter
Among these, only Brewer (1970) identified cor-
rectly the fact that such potentials are related to Adjust to the desired pH by adding 210-15
the length of the gel mold and should be ex- ml/liter bis(dimethy1arnino)-2-propanol
pressed as volts per linear centimeter of gel. We Electrode: Undiluted stock solution
find most published voltages to be at or beyond
the high end of applicability and improved reso- Gel: 1:19 dilution of stock solution
lution (as well as lab planning) can be gained This buffer was described by Clayton and Tretiak
with electrophoretic runs for longer duration at (1972).It may be optimal at pH 27.5.
lower voltages.

Borate (continuous), pH 8,6

Electrophoresis Tracking Dyc
Stock solution:
amaranth (0.25 M) boric acid 15.5 g/liter
brilliant blue G
ethanol Adjust to pH 8.6 with NaOH (pellets)
Hz0 Electrode: Undiluted stock solution
This stain was modified from a recipe formerly Gel: 1:9 dilution of stock solution
prepared by Gelman Sciences, Inc., Ann Arbor,
MI, U.S.A. It gives both blue and red markers. It This buffer was modified from Sackler (1966)who
used it at pH 8.8.
118 Chapter 4 / Murphy, Sites, Buth t3 Haufler

Borate (discontinuok~s) which is a slight modification of that described by

Ridgway et al. (1970).This buffer will often cause
Electrode: gels to separate as the citric acid buffer front
(0.30 M) boric acid 18.6 g/liter passes through the origin.
(0.03 M) sodium chloride 1.75 g/liter
Adjust to pH 8.0 with 2 N NaOH
Gel: (0.02M) boric acid 1.24 g/liter Stock solution:
Adjust to pH 8.6 with 2 N NaOH (0.214 M) potassium phosphate
dibasic (K2HP04) 37.3 glliter
This buffer was described by Brewer (1970).
(0.027 M) citric acid monohydrate 5.67 glliter
Adjust to pH 7.0
Histidine-C itrate (discesntinuous)
Electrode: Undiluted stock solution
Gel: 1:25 dilution of stock solution
(0.41 M) citric acid dihydrate
(trisodium salt) 20.6 g/liter This buffer was modified from Selander et al.
(1971), who called for 0.214 M potassium phos-
Adjust to pH 7.0 or 8.0 with HC1. phate dibasic but provided the measurement
Gel: (5 mM) L-histidine (g/liter) for this molarity of potassium phosphate
HC1 monohydrate 1.05 g/liter monobasic (KH2P04).If the latter is used, use 29.1
g/liter. Harris and Hopkinson (1976) described a
Adjust to pH 7.0 or 8.0 with NaOH. similar buffer using 0.245 M sodium phosphate
This buffer was described by Fildes and Harris monobasic and 0.15 M citric acid monohydrate
(1966), Brewer (1970), and Harris and Hopkinson adjusted within the range of pH 5.9-7.5; dilute the
(1976). The electrical current may drastically in- stock 1:39 for the gel.
crease after running for a few hours. These gels
should be monitored while running.

Stock solution:
(0.90 M) Tris 9.0 g/liter
Stock solution A: (0.50 M) boric acid 0.9 g/liter
(0.19 M ) boric acid 11.8 &liter (0.02 M) disodium EDTA 6.7 glliter
(0.03 M) lithium hydroxide Adjust to pH 8.6 with NaOH (pellets)
(LiOH*H20) 1.26 glliter
Adjust to pH 8.1
Anode: 35 ml stock solution + 215 ml H 2 0
Stock solution B: (1:6 dilution)
(0.05 M) Tris 6.06 glliter Cathode: 50 ml stock solution + 200 ml H 2 0
(0.008 M ) citric acid monohydrate 1.68 glliter (1:4 dilution)
Adjust to pH 8.4 Gel: 1:19 dilution of stock solution
Electrode: Undiluted stock solution A This is a modification of the buffer described by
Boyer et al. (1963) referred to as EBT by F.R. Wil-
Gel: 1:9 mixture of stock solutions A:B, final
son et al. (1973) and Shaklee and Keenan (1986),
pH 8.3
and as TBE by Aebersold et al. (1987).Shaklee and
This discontinuous buffer is the lithium hydrox- Keenan (1986) recommended the use of 7.4 g/liter
ide buffer described by Selander et al. (1971), tetrasodium EDTA in this buffer.
Proteins: Isozyme Electrophoresis 119

Stock solution: H20

(0.50 M) Tris 60.6 g/liter This buffer system was described by B.J. Turner
(0.65 M) boric acid 40.2 g/liter (1973).
(0.02 M) disodium EDTA 6.7 g/liter
Adjust to pH 8.0
Electrode: Undiluted stock solution Stock solution:
Gel: 1:9 dilution of stock solution (0.687 M ) Tris 83.2 g/liter
These gels tend to be thick and are particularly (0.157 M) citric acid monohydrate 33.0 g/liter
difficult to aspirate, pour, and slice. Stock solu- Adjust to pH 8.0
tions are not suitable for long-term storage and
better results may be found using fresh solutions. Electrode: Undiluted stock solution
This is the TVB (Tris-versene-borate)buffer of Se- Gel: 1:29 dilution of stock solution
lander et al. (1971) and Siciliano and Shaw (1976).
Another version of this buffer is described by This is the continuous Tris-citrate I1 buffer of Se-
Brewer (1970): 0.21 M Tris, 0.15 M boric acid, and lander et al. (1971). D.E. Soltis et al. (1983) listed
6 mM disodium EDTA adjusted to pH 8.0 for the other modifications of the Tris-citrate buffers of
electrode and 21 mM Tris, 20 mM boric acid, and Shaw and Prasad (1970) including (1) 0.135 M
0.68 mM disodium EDTA adjusted to pH 8.6 for Tris, 0.032 M citric acid, pH 8.0, diluted 1:14 for
the gel. Werth (1985) described another system, the gel, (2) 0.135 M Tris, 0.017 M citric acid, pH
termed "salamander B" and attributed to S.I. 8.5, diluted 1:14 for the gel, (3) 0.223 M Tris, 0.086
Guttman, which uses the same stock solution and M citric acid, pH 7.5, diluted 1:27.5for the gel, and
concentrations for the electrode and gel buffers: 84 (4) 0.223 M Tris, 0.069 M citric acid, pH 7.2, di-
mM Tris, 7.9 mM boric acid, and 0.86 mM di- luted 1:27.5 for the gel. Other variations include
sodium EDTA adjusted to pH 9.1 with HC1. 0.13 M Tris, 0.043 M citric acid, pH 7.0, diluted
1:14 for the gel (Siciliano and Shaw, 1976), 0.094 M
Tris, 0.0235 M citric acid, pH 8.6, diluted 1:5 for
the gel (Harris and Hopkinson, 1976), and 0.22 M
Tris, 0.086 M citric acid, pH 5.8, diluted 1:27.5 for
Stock solution: the gel (Shaklee and Keenan, 1986).
(0.9 M) Tris 109 g/liter
(0.4 M) lithium hydroxide
(LiOHaH20) 16.8 g/liter Tris-@ifrateBII
(0.5 M) boric acid 30.9 g/liter Stock solution:
(0.1 M) EDTA free acid 29.2 g (0.75 M) Tris 90.8 g/liter
H20 to 1000 ml (0.25 M) citric acid monohydrate 52.5 g/liter
Adjust to pH 9.1 with NaOH Adjust to pH 7.0 with NaOH (pellets).
Electrode: Electrode:
stock solution Anode: 35 ml stock solution + 215 ml H 2 0
SucTose (1:6 dilution)
H20 Cathode: 50 ml stock solution + 200 ml H 2 0
Gel: (1:4 dilution)

stock solution 40 ml Gel: 1:19 dilution of stock solution

120 Chapter 4 / Murphy, Sites, Buth & Haufler

This buffer was described by Whitt (1970) and Adjust to pH 9.6

Rainboth and Whitt (1974).
Electrode: Undiluted stock solution
Gel: 1:9 dilution of stock buffer
This buffer was described by Harris and Hopkin-
Electrode: son (1976).
(0.30 M) boric acid 18.6 g/liter
(0.06 M) sodium hydroxide 2.4 g/liter
Adjust to pH 8.2
(0.30 M) boric acid 18.6 g/liter
(0.076 M) Tris 9.21 g/liter (0.06 M) sodium hydroxide 2.4 g/liter
(0.005M) citric acid monohydrate 1.05 g/liter
Adjust to pH 8.2
Adjust to pH 8.7
This is the discontinuous Tris-citrate or Poulik
buffer described by Selander et al. (1971). This (0.01 M) Tris
buffer will often cause gels to separate as the bo- Adjust to pH 8.5 using concentrated HC1
rate buffer front passes through the origin. Chip-
pindale (1989) modified this system by using 26.2 This discontinuous buffer was described by Se-
g/liter Tris to bring the pH of the gel buffer to 9.5; lander et al. (1971). Harris and Hopkinson (1976)
his modification is excellent for resolving other- recommended a continuous buffer modification
wise fuzzy systems. using 0.1 M Tris-HC1with a 1:4 dilution for the gel
or 0.3 M Tris-HC1 with a 1:14 dilution for the gel.
They recommended the use of this buffer in the
range of pH 8.6-9.6.
Stock solution:
(0.135 M) Tris 16.4 g/liter
(0.045 M) citric acid monohydrate 9.46 g/liter Stock solution:
(1.3 mM) disodium EDTA 0.44 g/liter
(0.10 M) Tris 12.1 g/liter
Adjust to pH 7.0 (0.10 M) maleic acid 11.6 g/liter
(0.01 M) disodium EDTA 3.36 g/liter
Electrode: Undiluted stock solution
(0.01 M ) MgC12 6H20 2.03 g/liter
Gel: 1:14 dilution of stock solution
Adjust to pH 7.4 with NaOH
This buffer is modified from Avise et al. (1975).A
modification of this system using different molar- Electrode: Undiluted stock solution
ities of the components (pH 5.7-8.6) and limiting Gel: 1:9 dilution of stock solution
the disodium EDTA to the gel buffer was de-
scribed by Harris and Hopkinson (1976). This buffer was described by Brewer (1970) and
Selander et al. (1971) at pH 7.6 and 7.4, respec-
tively. Brewer (1970) noted that some of the
reagents (e.g., EDTA) will not go into solution un-
til the NaOH is added. Harris and Hopkinson
Stock solution: (1976) recommended the use of this buffer in the
(0.1 M) Tris 12.1 g/liter range pH 6.5-7.4.
(0.0045M) disodium EDTA 1.5 g/liter