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USOO5292665A
United States P?t?llt [19] [11] Patent Number: 5,292,665
Hochstrasser et al. [45] Date of Patent: Mar. 8, 1994

[54] CATALYST FOR PREPARING 4,950,708 8/1990 Hochstrasser ..................... .. 524/728


POLYACRYLAMIDE GEL WHICH OTHER PUBLICATIONS
INIPROVES THE DETECTION OF
BIOMATERIALS BY SILVER STAINING Artoni et al., Fractionation Techniques in a Hy
1751 Inventors: Denis F. Hochstrasser, Geneva, dro-Organic Environment Analytical Biochemistry
Switzerland; Carl R. Merril, 137, 1984, pp. 420-428.
Rockville, Md. Aldrich, p. 620, catalog No. D17,930-2.
Kirk—0thmer, Encyclopedia of Chemical Technol
[73] Assignee: The United States of America as ogy, 3rd Edition, vol. 22, 1983, pp. 978-982.
represented by the Department of Hochstrasser et al., Development of Polyacrylamide
Health and Human Services, Gels . . . Staining, Analytical Chemistry 173, 412-423
Washington, Dc.
(1988).
[21] Appl. No.: 849,344 US. Dept. of Commerce, NTIS publication
PB88-179809 May 17, 1988. ,
[22] Filed: Mar. 11, 1992
US. Dept. of Commerce, NTIS publication
Related US. Application Data PB88-2l2758 Apr. 7, 1988.
[63] Continuation of Ser. No. 323,851, Mar. 15, 1989, aban Primary Examiner-James C. Housel
doned. Assistant Examiner-Long V. Le
Attorney, Agent, or Firm-Leydig, Voit 8L Mayer
[51] Int. Cl.5 ........................................... .. G01N 21/77
[52] US. Cl. .................................... .. 436/86; 436/174; [57] ABSTRACT
436/905; 204/180.l; 204/l82.8 A polyacrylamide gel comprising acrylamide and dia
[53] Field 01' Search ................. .. 436/86, 79, 174, 905; crylylpiperazine. A method for providing a polyacryl
1204/1801, 182.6, 182.8; 524/521, 728; 525/279 amide gel comprising employing a catalyst system
[56] References Cited which comprises dimethylpiperazine, sodium thiosul
U.S. PATENT DOCUMENTS fate or a mixture thereof and ammonium or potassium
persulfate. The use of the gel as the matrix in a silver
4,189,370 2/ 1980 Boschetti ........................... .. 204/299
staining procedure for the detection of biomaterials,
4,421,915 12/1983 Ponticello et al. .. 544/387
4,555,490 11/1985 Merril ......................... .. 436/86
provides for the obtainment of reduced background
4,654,132 3/1987 Takagi et al. 204/l82.8 staining.
4,874,490 10/1989 Hochstrasser . . . . . . . . . . .. 204/ 182.1

4,912,011 3/1990 Yamamoto et al. .............. .. 430/138 15 Claims, 1 Drawing Sheet


US. Patent Mar. 8, 1994 5,292,665

11
10

kiwnda-40o
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FIG. 1

FIG. 2
5,292,665
1 2
patent does not stain proteins or nucleic acids in thin
CATALYST FOR PREPARING membranes such as cellulose nitrate.
POLYACRYLAMIDE GEL WHICH HWPROVES Coomassie blue stain, the most commonly employed
THE DETECTION OF BIOMATERIAIS BY protein stain, takes hours to perform and it lacks the
SILVER STAINING sensitivity to detect proteins present in low concentra
tions in biological fluids or tissues. Sensitivities
This is a continuation of application Ser. No. achieved with heavy metal stains or ?uorescent stains,
07/323,851, ?led Mar. 15, 1989, now abandoned. on the other hand, were found to be less than, or at best,
equivalent to Coomassie blue (about 10 ng of protein).
BACKGROUND OF THE INVENTION 10 Merril et al., Electrophoresis, 1982, pp. 327-342. Re
1. Field of the Invention cently, more than a hundred-fold increase in sensitivity
The present invention relates to a novel polyacryl over that obtained with Coomassie staining was
amide gel and process for the preparation thereof. The achieved by adapting a histological silver tissue stain for
polyacrylamide gel improves silver stain methods for use with polyacrylamide gels. de Olmos, Brain, Behav.
detecting biomaterials, such as proteins, polypeptides, 15 EvoL 2, 313-237 (1969), Switzer, et a1. Anal. Biochem.
nucleic acids, and the like, which had been separated by 98, 231-237 (1979), Merril, et al. Proc. Natl. Acad Sci.
electrophoresis procedures. Particularly, the novel USA. 76, 4335-4339 (1979). This stain could detect as
polyacrylamide gel provides for the detection of prote little as a tenth of a nanogram of protein and an image
ins by ammoniacal silver staining with reduced back could be achieved in less than 6 hours.
ground staining. 20 There are a number of known methods useful in stain
2. Discussion of Related Art ing proteins which utilize silver. For example, L. Kere
In the past, various methods have been used for pro nyi, et al., Clin. Chim. Acta 38, 465-467 (1972), de
tein analysis. Such methods include the Kjehdahl scribes a method for demonstrating proteins in electro
method, a colorimetric method, and others. In more phoretic, immunoelectrophoretic and immunodiffusion
recent times, electrophoresis techniques were em 25 preparations, whereby the preparations are treated with
ployed. For example, a protein sample was ?xed onto a potassium ferrocyanide, which is transformed during
matrix, such as a polyacrylamide gel, and subjected to development into silver ferrocyanide and then into
electrophoretic separation. This separation is accom colloidal silver grains. The physical developer contains
plished by exposing the sample to an electric ?eld anhydrous sodium carbonate, ammonium nitrate, silver
which causes the various components of the sample to
30 nitrate, tungstosilicic acid and formalin, and the protein
. migrate at different rates within the matrix. These differ in the preparations stain dark brown with a page gray
ent rates are dependent on the charge of the individual
background.
components, as well as on the other physical and chemi R. C. Switzer, et al., Anal. Biochem. 98, 231-237
cal properties thereof. Following the migration, a cer 35 (1979), and C. R. Merril, et al., Proc. Natl. Acad. Sci.
tain migration pattern is formed. Various methods for U.S.A., 76 No. 9, 4335-4339 (1979), describe a silver
de?ning such migration patterns have developed, such stain technique for detecting proteins and peptides in
polyacrylamide gels which is a modi?cation of de 01
as visual determination methods. Radioautography and mos’ neural, cupric-silver stain. The procedure consists
staining are among the procedures which are included of ten steps and utilizes an aqueous solution of silver
within the visualization methods. nitrate and cupric nitrate and involves treatment with a
Radioautography is conducted, for example, with diamine solution, which is known to sometimes form an
intrinsically labelled proteins produced from substrates explosive silver amide complex. The proteins stain as
containing radioactive labelled amino acids. See article dark spots on a darkened background.
by P. H. O’Farrell, in J. Biol. Chem, Vol. 250, pp. B. A. Oakley, et al., Anal. Biochem. 105, 361-363
4007-4021 (1975). Moreover, extrinsic radioactive la 45 (1980), simpli?ed the above procedure of Switzer, et al.,
belling techniques, such as iodination, can alter the by reducing the number of steps involved to six and also
electrophoresis properties of proteins, and the isotope is reducing the amount of silver required without dimin
not necessarily uniformly linked to each protein and ishing the sensitivity of the technique. However, the
non-selectively distributed among all polypeptide com manner in which the proteins stain was not changed,
ponents of the mixture. 50 i.e., dark stain on a darkened background.
Direct iodination of separated proteins in polyacryl A further modi?cation of the Switzer, et al., proce
amide gels has been reported by Edler et a]. (see the dure was made by R. C. Allen, Electrophoresis 1, 32-37
article by J. H. Edler, R. A. Peckett, II, J. Hampton, (1980), who increased the sodium to ammonium ion
and R. A. Lerner in the J. Biol Chem., Vol. 252, pp. ratio, which resulted in increased silver deposition.
6510-6515 (1977)). This technique is quite useful when 55 C. R. Merrill, et al., Anal. Biochem, 110, 201-207
used to study radioactive peptides after tryptic diges (1981), modi?ed and simpli?ed the above procedure of
tion, but it has been found that some lots of acrylamide Kerenyi, et al., adapting it to acrylamide gels.
contain a contaminant which also becomes radioiodin D. Goldman, eta1., Clin. Chem. 26 No. 9, 1317-1322
ated, and this negates its utility for radioautography. (1980), report that when using a procedure essentially
Even though radioautography is a powerful visual 60 the same as that of Merrill, et al. (PNAS, 1976), and
ization tool, it has certain disadvantages. For example, it Switzer et al., (Anal. Biochem., 1979), proteins from
is slow and complex. Also, it involves modi?cations to samples of cerebral spinal ?uid stained in shades of
the proteins prior to electrophoresis. yellow, red and blue.
One of the methods known for visualization of pro C. R. Merrill, et al., Science 211, 1437-1438 (1981),
tein is described in U.S. Pat. No. 4,405,720. The stain describe a silver stain procedure for proteins separated
described in this patent requires the use of three solu by two-dimensional gel electrophoresis, which requires
tions and it takes a minimum of about 30 minutes to treatment with potassium dichromate and nitric acid
perform. Furthermore, the technique described in said prior to staining with silver nitrate followed by washing
5,292,665
3 4
and then immersion in an image developer containing of specimens with periodic or hydrochloric acid, thi
formalin and sodium carbonate. There is no indication ocarbohydrazide or thiosemicarbazide, and silver me
of color development with this stain procedure. thenamine. The technique, when using periodic acid,
Poehling and Neuhofl‘, Electrophoresis 1981, 2, provides an excellent stain to evaluate glycomac
141-147, describe a silver stain suitable for acrylamide romolecules and ?brovascular tissue and to conduct a
gels of 0.5 to 1 mm thickness which requires a pretreat broad spectrum of staining procedures for all modes of
ment with glutardialdehyde under controlled tempera microscopy. Use of hydrochloric acid facilitates evalua
tures prior to staining with a diamine solution. tion of cell nuclear DNA and chromatin.
Marshall and Latner, Electrophoresis 1981, 2, U.S. Pat. No 4,695,548 describes gel inserts compris
228-235, describe a silver stain method which requires a ing a solidi?ed liquid, such as agarose, suitable for use in
tratment with paraformaldehyde and sodium cacodyl an electrophoretic method, lysed cells entrapped within
ate prior to staining with a modi?ed diamine solution a matrix formed by the solidi?ed liquid and macromole
wherein methylamine is substituted for ammonium hy cules, such as DNA or intact chromosomes derived
droxide. Ochs et al., Electrophoresis 1981, 2, 304-307, from the lysed cells, may be advantageously used in
and Sammons and Adams, Electrophoresis 1981, 2, electrophoretic separations. The gel inserts are placed
135-145, describe a silver stain procedure of which the directly in a suitable support medium and subjected to
present invention is a modi?cation. one or more electric ?elds to separate the macromole
With the exception of the 1980 Goldman et al. proce cules.
dure and the method of Sammons and Adams, all of the US. Pat. No. 4,468,466 describes a silver stain
silver stain techniques described above only stain prote 20 method for protein in gels utilizing treatment with a
ins in varying shades of brown or black.
reducing agent followed by treatment with a silver salt
US. Pat. No. 4,416,998 describes a silver stain proce
dure, wherein a substance capable of binding silver is and actuating irradiation, the improvement comprising
treated with a glutaraldehyde solution, an aqueous sil
the use of a reducing agent consisting essentially of
ver salt solution, a reducing solution and an aqueous
dithiothreitol in an amount effective to stain the protein
carbonate or sulfate solution. The procedure also ena but keep background staining to a
bles one to stain a variety of substances, including pro As illustrated above, silver staining methods, which
tein, in varying shades of color. employ polyscrylarnide gel for detecting biomaterials,
US. Pat. No. 4,582,808 describes a silver staining are widely used. However, unacceptable background
method comprising pretreating a carrier, such as a poly staining drawbacks are associated with each of these
acrylamide gel, with an alcoholic solution containing illustrative methods, including the method of US. Pat.
polyethylene glycol or polyoxyethylene alkylphenol, No. 4,468,466, which is mentioned as keeping back
followed by treating the pretreated carrier with a solu ground staining to a
tion of silver nitrate. This method is disclosed as having Recent observations concerning the mechanisms of
a shortened operation time and an improvement in the silver stains have led to the development of a polyacryl
reproducibility of staining. amide gel, which does produce very little, if any, back
U.S. Pat. No. 4,555,490 describes a method using ground staining. The key observations, which permitted
light ("photodevelopment”) to develop a metallic silver the development of this gel, are: the essential nature of
image of biopolymers, particularly nucleic acids and basic amino acids containing sulfur in the detection of
proteins separated on polyacrylamide gels, whereby it is 40 peptides by the silver staining reaction; and evidence
possible to visualize protein and nucleic acid patterns that the active groups in the basic amino acids, the
within 10 minutes after electrophoretic separation. This imidazole, guanidine and amino groups, or the sulfur
“photodevelopment” method requires only two solu groups in the sulfur containing amino acids, require
tions: a solution to “?x” the proteins and a solution cooperative effects. That is, they function poorly when
containing silver ions, which produces an image when 45 they are isolated in a polymer, but if two or more basic
exposed to light. This type of protein stain has achieved amino acids of sulfur containing amino acids are in close
a sensitivity of about 0.5 ng of protein. DNA separated proximity, then a good staining reaction will occur.
on polyaorylamide may also be visualized with this These studies on the mechanisms of silver stains led the
stain. present inventors to determine that the amide groups in
US. Pat. No. 4,575,452 describes a method and kit for 50 methylene-bisacrylamide crosslinking agent might be
the optical detection of proteins and nucleic acids in a responsible at least partially for the background found
matrix, such as polyaorylamide electrophoresis gels. with the silver stains. Methylene-bisacrylamide contains
The method comprises ?xing the proteins and nucleic two amide groups, which are separated by a single
acids in the matrix using aromatic sulfonic acids having carbon. in this study, the present inventors have demon
tertiary amines capable of forming coordination com strated the role of these amide groups in the formation
plexes with silver ion. of the background stain by studying the silver stain
U.S. Pat. No. 4,672,043 describes a method for deter reaction in gels containing varying ratios of acrylamide
mining macromolecules in polyacrylamide gels com to the methylene-bisacrylamide crosslinking agent. By
prising the steps of forming a latent stain image by nu utilizing different crosslinking agents, the present inven
cleating the macromolecules in the gel with a palladium tors have demonstrated that the appearance of back
tetramine salt and developing the latent stain image by ground staining depends mainly on the presence of and
treating the gel with a physical developing solution the position of the amido groups in the crosslinking
comprising dimethylamine borane and a transition agents. It depends also on the presence of other groups
metal salt. The improvement comprises contacting the in the crosslinker or the acrylamide chain.
developed latent stain image with a l-phenyl-Z-tetrazo
line-5-thione or a salt of l-phenyl-ll-l-tetrazole-S-thiol. SUMMARY OF THE INVENTION
US. Pat. No. 4,690,901 describes a staining technique An object of the present invention is to provide a
for specimens, which involves the sequential treatment novel polyacrylamide gel.
5,292,665
5 6
Another object of the present invention is to provide 0: no diamine used; the concentration of persulate
a method for producing the polyacrylamide gel. was increased.
A further object of the present invention is to provide This gel demonstrated that the lowest background
an ammoniacal silver staining biomateiial detection staining could be achieved by polymerizing at a poly
method with reduced background staining. acrylamide gel with diacrylylpiperazine (PIP) as cross
The present invention relates to a polyacryl amide gel linking agent, with dimethylpiperazine and ammonium
comprising acrylamide and diacrylylpiperazine. Prefer ‘or potassium persulfate as the catalytic system. Back
ably, the weight ratio of acrylamide to diacrylylpipera ground staining was further minimized if the catalytic
zine (PIP) is about 15 to 0.4. system contained sodium thiosulfate.
The present invention further relates to a method for Despite extensive washing procedures and/or pre
preparing a polyacrylamide gel comprising polymeriz electrophoresis of the gel, the background staining was
ing acrylamide with diacrylylpiperazine in the presence profoundly affected by the diamine, the salts and the
of a catalyst system which comprises dimethylpipera oxido-reducing catalyst. This observation provides indi
zine or preferably sodium thiosulfate and ammonium rect evidence that these compounds may be incorpo
persulfate or potassium persulfate. More preferably, the rated in the structure of the gel. Small ions should have
catalyst system comprises a mixture of dimethylpipera been removed by preelectrophoresis and washing pro
zine, sodium thiosulfate and ammonium persulfate or cedures. The diamine bases and anions are probably
potassium persulfate. incorporated into the gel matrix. The lack of an effect
Yet further, the present invention relates a silver stain with the cations suggest that they are not incorporated
method for the detection of biomaterials with reduced into the gel under the basic conditions utilized in these
background staining, the improvement which com experiments.
prises the use of a polyacrylamide gel comprising acryl DETAILED DESCRIPTION OF THE
amide and diacrylylpiperazine as the matrix. Preferably, INVENTION
the silver stain method is an ammoniacal silver stain
method and the biomaterials are proteins.
25 The above objects and advantages in accordance
with the present invention are achieved by providing a
In a more preferred silver stain method for the detec
tion of biomaterials with reduced background staining,
novel polyacrylamide gel. The polyacrylamide gel is
comprised of acrylamide and diacrylylpiperazine. Pref
the improvement comprises the use of a polyacrylamide erably, the weight ratio of the acrylamide to diacrylyl
gel produced by polymerizing acrylamide with dia 30 piperazine comprised in the polyacrylamide gel is about
crylylpiperazine as the matrix in the presence of a cata 30 to 0.8. The novel polyacrylamide gel is formed by a
lyst system, which comprises dimethylpiperazine, so method comprising polymerizing acrylamide with the
dium thiosulfate or a mixture thereof and ammonium crosslinker, diacrylylpiperazine, in the presence of a
persulfate or potassium persulfate. catalyst system which comprises dimethylpiperazine or
BRIEF DESCRIPTION OF THE DRAWINGS 35 preferably sodium thiosulfate and ammonium or potas
sium persulfate. More preferably, the catalyst system,
FIG. 1 is a photograph of an electrophorasis gel, comprises a mixture of dimethylpiperazine, sodium
which illustrates the effects of different diamine cata thiosulfate and ammonium persulfate or potassium per
lysts on silver staining in polyacrylamide gels. A vol sulfate. Preferably, the catalyst system is present in an
ume of 150 pL of ammonium persulfate solution was 40 amount of about 0.8 to 16 mg/g based on the total
used in all gel bands, except for the band containing weight of the acrylamide and diacrylylpiperazine. In
l,4-diazabicyclo(2,2,2)octane which required 800 pL of further accordance with the foregoing, the employment
ammonium persulfate solution for an identical polymer of the novel polyacrylamide gel as the matrix in an
ization rate. The varying amounts of diamines, were ammoniacal silver staining procedure-for the detection
chosen to obtain similar rate of polymerization. It was 45 of biomaterials, particularly proteins, provides for the
impossible to produce a gel with equivalent mechanical obtainment of reduced background staining.
properties with triethylamine, 1,3-diaminopropane or The formation of background staining has limited the
l,5-diazabicyclo(4,3,0)non-5ene. The polymerization sensitivity, reproducibility, and quantitative analyses, of
was much poorer with these three reagents. All the most polyacrylamide gel silver staining methods. It was
bands were polymerized with diacrylylpiperazine (PIP) found that methylenebisacrylamide (BIS), the most
as the crosslinking agent except band one which was commonly used gel crosslinker, was partially responsi
polymerized with methylenebisacrylamide (BIS) and ble for this background stain, when polyacrylamide gels
TEMED to illustrate the current commonly employed were stained with ammoniacal silver nitrate. Gels poly
gel polymerization system. merized with BIS appear black and opaque after a pro
FIG. 2 is a photograph of an electrophoresis gel, longed development time. The development of a new
which illustrates the effects of different “catalysts” and crosslinking agent, diacrylyl~piperazine (PIP) pro
adjunct compounds on silver staining in polyacrylamide foundly diminished this background staining with am
gels. moniacal silver stain. However, after a prolonged de
APS: 1 g/L velopment period, a yellow background still appeared.
KPS: 0.67 g/L The residual yellow background which was obtained
All other salt solutions: 0.75 g/L with the PIP crosslinked gels, appeared to be related to
TEMED (N,N,N'N'-tetramethylethylenediamine): the catalyst used for the polymerization of the gel.
3.3 mL/L Many catalyst systems have been suggested in the
DMPIP (IA-dimethylpiperazine): 20 mL/L past. The most commonly used contain: ammonium
BIS: methylene-bisacrylamide 0.8 g/ 100 mL stock 65 persulfate (APS) or potassium persulfate (KPS), ribo?a
solution vin (RFN) and N,N,N'N'-tetramethylethylenediamine
PIP: diacrylyl-piperazine 0.1 g/ 100 mL stock solu (TEMED). The catalytic systems can be divided in two
tion general groups: a) one in which free radicals are gener
5,292,665
7
ated by photocatalysis and mother b) in which free in controlling the rate of polymerization have been
radicals are generated by oxydo-reduction reactions. reported for this system. The acetic acid, which was
a) The agent most widely used as photo-catalyst, often used in the staining procedure, may also react
RFN, was suggested for vinyl group polymerization withrthe hydrogen peroxide to form peracetic acid,
four decades ago. Its photo-decomposition can be en 5 which has a tendency to attack the Plexiglas electro
hanced by cinnamyl alcohol or by the dye methylene phoretic apparatuses.
blue-triethanolamine. The use of ribo?avin-5'-phos None of the systems outlined above has been found to
phate (RFP) has also been proposed. The photodecom be optimal for the polymerization of gels, when silver
position of these ribo?avin molecules produces the free stain techniques are used to detect proteins. Photocata
radicals. lytic agents have been avoided because of the dif?cul
Ribo?avin molecules have several disadvantages: ties in obtaining reproducible polymerization in gel
they cannot be used at acidic polymerization conditions casting chambers.
(pH 3), when the catalyst system has to be optimized for The diamine-persulfate system produces a yellow
the gel and buffer system considered; they require background stain with ammoniacal silver a?er a pro
highly reproducible UV or visible light illumination to 15 longed development period.
get reproducible gels especially in a casting chamber The current study presents the results of a number of
when multiple gels are polymerized Uranyl nitrate can diamine-salt catalytic systems on the polymerization
be used as a photopolymerin'ng agent at a low pH, but and the silver staining of gels. A new catalytic system,
the hazards associated with its uses (radioactivity, possi which further delays or even prevents the appearance
ble explosive material obtained), although small, are of a background stain with ammoniacal silver stain and
cumbersome. _ which, therefore, enhances protein detection, is de
b) The redox systems most commonly used are APS scribed.
or KPS and a diamine compound as an adjunct catalyst. Chemical structures of diamines used:
TEMED is the most commonly used diamine and occa
sionally, dimethylaminopropionitrile (DMPN) is uti
lized. It has been suggested to replace the diamine with
sodium sul?te or even silver nitrate. The redox system
most rarely used is hydrogen peroxide-ferrous sulfate
ascorbic acid, also called Fenton’s reagent. Difficulties

Nl-i2—CH2—CH1—Cl-l2-NH1 1,3-diaminopropane
CH3 S-dimethylaminopropylamine
CH3—N—CH;—CH2—CH1—NH
CH3 dimethylaminopropionitrile

4- Gig-CH l-piperidinepropionitrile
/

Gill-CH1
5. CH2_.CH CH3 dirnethylaminoethylmorpholine

CHz-CHZ

cm-n-cm-cm-rm; dim?hylelhylmdilmine
CH3
dimethylethylethylencdiamine
CH3
N,N,N'N'-tetramethyl
ethyleuediamine (TEMED)

dimethylpiperazine (DMPIP)

diazabicyclo(2,2,2)octane
5,292,665
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MATERIALS AND METHODS mL of solution B and 800 pL of 100 g/L diazabicy
clooctane solution.
Apparatus Polymerization is initiated sequentially by the addi
The model 175 chamber (Bio-Rad, Richmond, Calif.) tion of 150 p1 of ammonium persulfate stock solution
was employed for isoelectric focusing (IEF) separation. (10 g/ 100 ml of water) to each of the ten gel solutions.
Sodium dodecylsulfate (SDS) polyacrylamide gel elec In the actual procedure, the polyacrylamide gels are
trophoresis (PAGE) separation was performed in the prepared by ?rst preparing the following solution: 173
Protean II® chamber (Bio-Rad, Richmond, Calif). mL of Tris HCl solution (pH 8.8, 1.5M), 265 mL of
Power was supplied with a 3000/300xi power supply acrylamide/diacrylylpiperazine solution (30 g/0.8 g in a
(Bio-Rad, Richmond, Calif.) for IEF separation, and ?nal volume of 100 mL with deionized water) and 207.5
700 V 1.6 Amp HP power supply (Hewlett-Packard, mL of deionized water. Prior to degassing the solution,
Palo Alto, Calif.) for the SDS-PAGE separation. The 4 mL of thiosulfate solution (5 gm of 100 mL in deion
gels (l80X200X 1.5 mm) were cast either in the Protean ized water) is added; then 4 mL of dimethylpiperazine
II® casting chamber or on the stand-alone casting
and 5 mL of ammonium persulfate solution (10 gm in
device (Bio‘Rad, Richmond, Va.). 100 mL of deionized water) are added to initiate the
Reagents polymerization and form the gels. Each of the gels is
The following reagents were used: acrylamide, N,N' cast by gently pouring 2 m1 of each of the solutions in
methylene-bisarylamide, N,N,N’N’-tetramethyle sequential order between two glass plates separated by
thylenediarnine (TEMED), ammonium persulfate, tri 1.5 mm spacers, at 4 min. intervals. The stepwise com
ethylamine, 1,3-diaminopropane, 3-dimethyl-amino posite gel, a “zebra” gel is removed from between the
propylamine, l-piperidinepropionitrile, dimethyle glass plates 2 hr. later and stained as described in “Pro
thylenediamine, dimethylethylethylenediamine, 1,4 tein detection” discussed below.
dimethylpiperazine, piperazine, 4(2(dimethylamino)e
thyl)morpholine, l,5-diazabicyclo(4.3.0)non-5ene, 1,4 Studies with gels polymerized with different
diazabicyclo-(2.2.2)octane, Tris HCl, citric acid, oxydo-reducing agents and adjunct compounds
cholamidopropyldirnethylhydroxypropanesulfonate The same stock solutions A and B described above
(CHAPS), ampholytes 3.5-10 and 5-7 (source: LKB, are used. Stock solution S is prepared by dissolving 10
Broma, Sweden); potassium persulfate, sodium sulfate, g of ammonium persulfate in 100 mL of deionized wa
sodium sul?te, sodium hydrosul?te, sodium bisulfite, ter. Stock solution K isprepared by dissolving 30 g of
potassium metabisul?te, sodium thiosulfate, sodium potassium persulfate in 100 mL of deionized water. All
permanganate and magnesium sulfate. the other salt solutions (sodium sulfate, permanganate,
Procedure-Effects of Varying the Diamine bisul?te, etc.) are prepared by dissolving 5 g of the
Compounds 35 considered salt in 100 mL of deionized water. The gel is

Acrylamide stock solutions are prepared by dis cast by gently pouring 2 ml of each of the solutions
solving acrylamide and either N,N'~methylenebisac described below immediately after their preparation
rylamide or diacrylylpiperazine in water in the follow between two glass plates separated by 1.5 mm spacers.
ing amounts: stock solution M contained 30 g of acryl Solution 11 contains 2.5 mL of solution A, 10 pL of
amide and 0.8 g of methylene-bisacrylamide, stock solu TEMED and 30 PL of solution S; solution 12 contains
tion P contained 30 g of acrylamide and 1.0 g of dia 2.5 mL of solution B, 10 uL of TEMED and 30 PL of
crylylpiperazine. These stock solutions are each ad solution S; solution 13 contains 2.5 mL of solution B, 10
justed with deionized water to a final volume of 100 ml. ;LL of TEMED and 60 PL of solution K; solution 14
Tris HCl stock solution T (1.5M, pH 8.8) is prepared by 45 contains 2.5 mL of solution B, 60 pL of dimethylpipera
dissolving 181 g of Tris HCl in 1 L of deionized water zine (DMPIP) and 60 111.. of solution K; solution 15
and adjusting the pH to 8.8 with concentrated HCl. contains 2.5 mL of solution B, 60 ;,:.L of DMPIP and 30
A stock solution A is prepared for the polymerization pL of solution S; solution 16 contains 2.5 mL of solution
of the gels by mixing 16.5 mL of deionized water with B, 10 pL of TEMED, 50 uL of sodium sulfate solution
14.6 mL of Tris HCl stock solution T and 24 mL of and 100 pL of solution K; solution 17 contains 2.5 mL
scrylamide/BIS stock solution M. A stock solution B is of solution B, 10 p.L of TEMED, 50 pL of sodium
prepared for the polymerization of the gels by mixing permanganate solution and 100 PL of solution K; solu
16.5 mL of deionized water with 14.6 mL of Tris HCl tion 18 contains 2.5 mL of solution B, 10 pl. of
solution T and with 24 mL of acrylamide/PIP stock TEMED, 50 PL of sodium bisul?te solution and 100 uL
solution P. 55 of solution K; solution 19 contains 2.5 mL of solution B,
Solution 1 contains 5 mL of solution A and 20 pL of
TEMED; solution 2 contains 5 mL of solution B and
10 pL of TEMED, 50 pL of sodium hydrosul?te solu
150 p.L of diaminopropane; solution 3 contains 5 mL of tion and 100 uL of solution K; solution 20 contains 2.5
solution B and 100 pL of dimethyl-aminopropylamine; mL of solution B, 10 uL of TEMED, 50 pl. of potas
solution 4 contains 5 mL of solution B and 50 pL of 60 sium metabisul?te and 100 pL of solution K; solution 21
piperidinepropionitrile; solution 5 contains 5 mL of contains 2.5 mL of solution B, 10 PL of TEMED, 50 “L
solution B and 10 pL of dimethylethylenediamine; solu of magnesium sulfate and 100 1.1L of solution K; solution
tion 6 contains 5 mL of solution B and 10 pL of dime 22 contains 2.5 mL of solution B, 10 p.L of TEMED, 50
thylethylethylenediamine; solution 7 contains 5 mL of uL of sodium thiosulfate solution and 100 pL of solu
solution B and 10 PL of TEMED; solution 8 contains 5 65 tion K; solution 23 contains 2.5 mL of solution B, 10 p.L
mL of solution B and 60 “L of dimethylpiperazine; of TEMED and 50 pL of solution K; solution 24 con
solution 9 contains 5 mL of solution B and 400 pL of tains 2.5 mL of solution B, 600 pL of solution K, but no
dimethylaminoethylmorpholine; solution 10 contains 5 TEMED or DMPIP.
5,292,665
11 12
known alternative catalytic systems were suitable for
High Resolution Potential of Gels (2-DGE) the reasons outlined in the introduction.
Polymerized with the New Catalyst System. A number of amine and diamine organic bases were
Two-dimensional gel electrophoresis (2-DGE) offers tested as potential substitutes for TEMED. TEMED is
the greatest electrophoretic resolution currently avail 5 an organic base with two tertiary amines separated by
able. Catalyst systems are tested for their resolving two carbons. The ?rst compounds tested, triethylamine,
power in a high resolution 2-DGE system. The isoelec l,3—diaminopropane and dimethylaminoethylmorpho
tric focusing (IEF) and SDS-PAGE gels are prepared line, were poor catalytic agents. S-dimethylamino
according to publication, Hochstraaer, D.F., Harring propylamine and l-piperidinepropionitrile were better
ton, M., Hochstrasser, A.C., Miller, M.J., Merril, CR. catalysts, but they produced an even darker back
(1988), Anal. Biochem. vol. 173, p. 214-232, except for ground than TEMED. The presence of three carbons
the second dimension separation, where TEMED is between the two amines or the addition of an oxygen
replaced by DMPIP (6x the TEMED volume) and seemed detrimental for a good polymerization. The
sodium thiosulfate is added in the same proportion as in dimethyl-ethylenediamine family gave the best catalytic
the previous paragraph. Sample preparation, sample 15 system for the polymerization of the gels but they all
loading and running conditions are according to said produced almost the same yellow background as
publication. TEMED. From these results, it seemed that the best
diamine compound should have at least a tertiary amine,
_ Protein detection if possible two, separated by two carbons. 1,4-dimethyl
Silver stain detection of proteins is performed follow piperazine (DMPIP) is a diamine compound which
ing the procedures of publications, l-lochstrasser, D.F., contains two tertiary amine groups separated by two
Harrington, M., Hochstrasser, A.C., Miller, M..l., Mer carbons. Indeed, it was found to be a good catalyst, not
ril, CR. (1988), Anal. Biochem. vol. 173, p. 214-232, as potent as TEMED, but to give less background than
and Oakley, B. R., Kirsch, D.R., Morris, N.R. (1980), TEMED with ammoniacal silver nitrate. Its structure is
25 very similar to the crosslinker, diacrylylpiperazine, and
Anal. Biochem. 105, 361-363: After the separation of
proteins is completed, the gels are washed in water for not far at all from TEMED. In fact, if TEMED were
5 min., in ethanol: acetic acid: water (40:10:50) for 1 cleaved in the middle of the ethylene group into two
identical pieces, and if the resulting residues were in
hour, and then in ethanolzacetic acid:water (5 :5:90) verted and rebonded, one would obtain DMPIP. In
for; 3 h. or overnight. After a water wash of 5 min., the
contrast, DMPIP gave more background than TEMED
gels are then soaked for 30 min. in a 10% glutaralde with dichromate silver stain. .
hyde solution. The glutaraldehyde is removed by exten The rapid polymerization of gel solution containing
sive water washes, 3X 10 min. washes followed by highly puri?ed diacrylylpiperazine might be due par
4 X 30 min. washes. The ammoniacal silver nitrate solu tially to the catalytic effect of the crosslinker, itself.
tion is prepared by the slow addition of a solution con Diazabicyclo(2,2,2) octane did not give a good poly
taining 6 g of silver nitrate in 30 ml of deionized water merization, probably because of its tertiary structure.
to a solution containing 10 ml of ammonium hydroxide All the molecules tested produced a background
25%, 1.5 ml of ION sodium hydroxide and 160 ml of staining, each with a different color: orange for 1,3
water. The ?nal volume is adjusted to 750 ml with diaminopropane, yellow for the dimethylethylenedia
deionized water. The gels are soaked in the ammoniacal mine family, or brown for the propiontrile group. De
silver nitrate solution for 10 min., then washed 3X5 spite the me of DMPIP, a yellow background stain still
min. with water. The developing solution contained 0.1 appears after prolonged silver staining.
g of citric acid and 1 ml of formaldehyde/liter of deion Since no further reductions in background staining
ized water. The gels are developed for 5 min. or 20 min. could be achieved beyond that observed with DMPIP
in this solution and placed in a solution containing 50 ml 45 or TEMED, different salts were tested either to replace
acetic acid/l L water for 1 hour to stop the develop the organic bases and to be added to the persulfate or
ment. For storage purpose, the acetic acid solution is even to replace the persulfate. None of the salts tested
replaced by a glycerolzethanohwater (2:10:88) solution. (see FIG. 2) produced adequate polymerization if used
Results and Discussion alone. Only ammonium or potassium persulfate used
50 with another compound produced good polymerization
The purpose of this study was to measure the effect of and their effect on the background staining was identi
the catalyst system on silver staining in polyacrylamide cal (FIG. 2, #14-17). However, the addition of differ
gels polymerized with diacrylylpiperazine (PIP). It has ent anions dramatically modi?ed the background stain
been known for many years that catalyst systems have a from a yellow stain for sulfate to a dark brown stain for
major in?uence on the chemical and physical properties permanganate. These results provide evidence that the
of the gels. Depending on the buffer used and the pH molecules utilized in the catalytic system are bound
range chosen for the separation technique, different within the gel matrix.
catalytic systems have been utilized. For the polymeri The addition of sodium sul?te accelerated the poly
ration of multiple basic gels in a casting chamber, the merization process, and gels were polymerized with
use of TEMED and ammonium or potassium persulfate APS and sul?te without organic base. However, sul?te
has been the most convenient and commonly employed increased the background staining more than TEMED
catalyst system so far. However, this system has been or DMPIP. None of the other salts tested could replace
shown to create a yellow background stain with silver the organic base. The addition of thiosulfate slowed
(see FIG. 1). Both TEMED and persulfate produce this down the polymerization, but it totally eliminated the
background despite extensive gel washes and pre-elec 65 appearance of any background during prolonged devel
trophoresis (FIG. 1). These ?ndings suggest that those opment times with cooled developer solution, despite
molecules are either bound to or entrapped into the gel the use of persulfate and TEMED or DMPIP (FIG. 2,
or modify the polymer during polymerization. No other #21). Polymerization of slab gels, utilized in the second
13
5,292,665
14
dimension of 2-dimensional gel electrophoresis, with -continued
DMPIP or TEMED, persulfate and thiosulfate, re Diamine Compound(s) name Crosslinker
suited in separation of proteins with no modi?cation of
25. I KPS (6 g/L) PIP
the apparent relative molecular mass. The two-dimen
(Numbers 12 to 25 correspond to Lanes 12 w 25, rapeetively.)
aional gel electrophoresis picture of a plasma sample APS: 1 g/L
showed an increased number of spots (5%) secondary KPS: 0.67 g/L
All other salt solutions: 0.75 g/L
to the prolonged development time without any appar TEMED (N,N,N'N'-tetramethylethylenediamine)z 3.3
ent background. DMPIP (dimethylpipaazine): 20 mL/L
FIG. 1: Effects of different diamine catalysts on silver BIS: methyluae-biaacrylamide 0.8 woo in]. stock solution
PIP: diacrylyl-piperazine 0.! woo in]. stock aolution
staining polyacrylamide gels. Ozaodiamineuedtheconoeutrationofpermlt'ate'niaeruaed.

This gel demonstrated that the lowest background


Compound name Volume (FL)
15 staining could be achieved by polymerizing a polyacryl
1. N,N,N'N’-tetramethylethylenediamine 20 amide gel with diacrylylpiperazine (PIP) as crosslinking
agent, with dimethylpiperazine and ammonium or po
tassium persulfate as the catalytic system. Background
staining is further if the catalytic system
N,N-dimethyl-ethylenediamine 20 contains sodium thiosulfate.
N,N-dimethyl-N'.ethyl-ethylenediamine Despite extensive washing procedures and/or pre

F52so0w-.a“! N,N,N'N'-tetramethyl-ethylenediamine
(TEMED)
1,4-dimethylpiperazine (DMPIP)
4-[2-(dimethylamino)ethyl]morpholine
l,4-diazabicycl0(Z,2,2)octane (DABCO)
1,4-dimethylpiperazine (DMPIP)
sa§'s‘é electrophoresis of the gel, the background staining is
profoundly affected by the diamine, the salts and the
oxido~reducing catalyst. This observation provides indi
25 rect evidence that these compounds may be incorpo
rated into the structure of the gel. Small ions should
have been removed by pre-electrophoresis and washing
(Numbers 1 to II eorreapondtoLanes l to 11, respectively.) procedures. The diamine bases and the anions are prob
ably incorporated into the gel matrix. The lack of an
(Numbers 1 to 11 correspond to Lanes 1 to 11, respec 30 effect with the cations suggest that they are not incor
tively.) porated into the gel under the basic conditions utilized
A volume of 150 pL of ammonium persulfate solution in these experiments \
is used in all gel bands, except for the band containing In conclusion, it was previously determined that the
use of crosslinking agents without amide groups or with
l,4-diazabicyclo(2,2,2)octane, which required 800 pL 35 amide groups, that are substituted twice, such as dia
of ammonium persulfate solution for an identical poly crylylpiperazine, prevented the rapid appearance of a
merization rate. The varying amounts of diamines are background stain and reduced the total background
chosen to obtain similar rates of polymerization. It is after prolonged staining with ammoniacal silver. The
impossible to get a gel with equivalent mechanical addition of thiosulfate in “SDS” slab gels to DMPIP or
4-0
properties with triethylamine, 1,3-diaminopropane or TEMED and APS totally prevents the appearance of
l,5-diazabicyclo(4,3,0)non-5ene. The polymerization is background staining with ammoniacal silver nitrate
much poorer with these reagents. All the bands are during a prolonged development time and profoundly
polymerized with diacrylypiperazine (PIP) as the cross reduces the appearance of background staining with
dichromate silver staining. This catalytic system en
linking agent, except band one, which is polymerized 45 hances silver stain protein spot de?nition.
with methylenebisacrylamide (BIS) and TEMED to The invention being thus described, it will be obvious
illustrate the currently commonly employed gel poly that the same may be varied in many ways. Such varia
merization system. tions are not to be regarded as a departure from the
FIG. 2: E?‘ects of different catalysts and adjunct 50 spirit and scope of the present invention, and all such
compounds on silver staining in polyacrylamide gels. modi?cations as would be obvious to one skilled in the
art are intended to be included within the scope of the
following claims.
Diamine Compound(s) name Crosslinker What is claimed:
l2. TEMED Ammonium persulfate BIS 55 1. A method for preparing a polyacrylamide gel com
(APS) prising polymerizing acrylamide with diacrylylpipera
l3. TEMED APS PIP zine in the presence of a catalyst system which com
l4. TEMED Potassium persulfate PIP prises dimethylpiperazine in combination with ammo
(KPS) nium persulfate or potassium persulfate.
l5. DMPIP KPS PIP 2. The method according to claim 1, wherein said
l6. DMPIP APS PIP
l7. TEMED KPS + Sodium sulfate PIP
catalyst system further comprises sodium thiosulfate.
l8. TEMED KPS + Sodium permanganate PIP
3. The method according to claim 2, wherein said
19. TEMED KPS + Sodium bisul?te PIP acrylamide and said diacrylylpiperazine are present in a
20. TEMED KPS + Sodium hydroaul?te PIP weight ratio of about 15 to 0.4.
21. TEMED KPS + Potassium metabisul?te PIP 65 4. The method according to claim 2, wherein said
22. TEMED KPS + Magnesium sulfate PIP acrylamide and said diacrylylpiperazine are present in
23. TEMED KPS + Sodium Thiosulfate PIP an amount of about 13 g and about 0.4 g, respectively,
24. TEMED KPS PIP per 100 ml of water.
5,292,665
15 16
5. The method according to claim 2, wherein said prising a matrix of arylamide with diacrylylpiperazine
catalyst system is present in an amount from about 0.8 polymerized in the presence of a catalyst system which
mg to about 16 mg per gram of the total weight of said comprises dimethylpiperazine in combination with am
acrylamide and said diacrylylpiperazine. monium persulfate or potassium persulfate.
6. The polyacryalrnide gel produced by the method 12. The method according to claim 11, wherein said
according to claim 1. catalyst system further comprises sodium thiosulfate.
7. The polyacrylamide gel produced by the method 13. The method according to claim 12, wherein said
according to claim 2. acrylamide and said diacrylylpiperazine are present in a
8. The polyacrylamide gel produced by the method weight ratio of about 15 to 0.4.
according to claim 3. 14. The method according to claim 12, wherein said
9. The polyacrylamide gel produced by the method acrylamide and said diacrylylpiperazine are present in
according to claim 4. an amount of about 13 g and about 0.4 g, respectively,
10. The polyacryalmide gel produced by the method per 100 ml of water.
according to claim 5. 15. The method according to claim 12, wherein said
11. An improved silver stain method utilizing a poly 15 catalyst system is present in an amount from about 0.8
acrylamide gel for the detection of biomaterials with mg to about 16 mg per gram of the total weight of said
reduced background staining, wherein the improve acrylamide and said diacrylylpiperazine.
ment comprises the use of a polyacrylamide gel com

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