Chapter 6

SDS Polyacrylamide Gel

Electrophoresis of
Proteins

B. J. Smith
Institute of Cancer Research, Chester Beatiy
Laboratories, Royal Cancer Hospital,
Fulham Road, London, United
Kingdom

Introduction
Probably the most widely used of techniques for
analyzing mixtures of proteins is SDS polyacrylamrde gel
electrophoresrs In this technique, proteins are reacted
with the anionic detergent, sodmm dodecylsulfate (SDS,
or sodium lauryl sulfate) to form negatively charged com-
plexes. The amount of SDS bound by a protein, and so the
charge on the complex, IS roughly proportional to Its size.
Commonly, about 1.4 g SDS IS bound per 1 g protein, al-
though there are exceptions to this rule The protems are
generally denatured and solubrlrzed by their bindmg of
SDS, and the complex forms a prolate elrpsoid or rod of a
length roughly proportionate to the protems molecular
weight Thus, proteins of either acidic or basic pl form

41
42 Smith

negatively charged complexes that can be separated on the

bases of differences in charges and sizes by electrophore-
sis through a sieve-like matrix of polyacrylamide gel.
Thus is the basis of the SDS gel system, but it owes its
popularity to its excellent powers of resolution that derive
from the use of a stacking gel. This system employs the
principles of isotachophoresrs, which effectively concen-
trates samples from large volumes (within reason) into
very small zones, that then leads to better separation of
the different species. The system IS set up by making a
stacking gel on top of the separating gel, which IS of a
different pH. The sample is mtroduced to the system at
the stackmg gel. With an electric field applied, ions move
towards the electrodes, but at the pH prevarlmg in the
stacking gel, the protein-SDS complexes have mobrlitres
intermediate between the Cl- ions (present throughout
the system) and glycinate ions (present in the reservoir
buffer). The Cl- ions have the greatest mobility. The fol-
lowing larger ions concentrate into narrow zones in the
stacking gel, but are not effectively separated there. When
the movmg zones reach the separating gel, their respec-
tive mobilities change in the pH prevarlmg there and the
glycmate ion front overtakes the protein-SDS complex
zones to leave them in a uniformly buffered electric field to
separate from each other according to size and charge.
More detailed treatments of the theory of rsotachophoresis
and electrophoresis generally are available u-r the literature
(e.g., 1).
The system of buffers used in the gel system de-
scribed below is that of Laemmli (2), and IS used in a
polyacrylamide gel of slab shape. This form allows simul-
taneous electrophoresrs of more than one sample, and
thus IS ideal for comparatrve purposes.

Materials
1. The apparatus required is available commercially or can
be made m the workshop, but generally conforms to
the design by Studier (3) The gel IS prepared and run
m a narrow chamber formed by two glass plates sepa-
rated by spacers of perspex or other suitable material,
SDS Gels 43

as shown m Fig. 1. The spacers are longer than the

glass plates (so that they are easy to remove), are about
1 cm wide, and are of any thickness (say, 0.5-l mm for
a thin gel). The sample wells into which samples are
loaded are formed by a template comb that extends
across the top of the gel and is of the same thickness as
the spacers Typically, the teeth on this comb will be
1 cm long, 2-10 mm wide, and separated by about 3
mm. The chamber is sealed with white petroleum jelly
(Vaseline). A dc power supply IS also required.
2 Stock solutions. Chemicals should be analytical reagent
(Analar) grade and water should be distilled. Stock so-
lutions should all be filtered. Cold solutions should be
warmed to room temperature before use.

Fg. 1. The constructron of a slab gel, showmg the posltlons of

the glass plates, the spacers, and the comb.
44 Smith

(1) Stock ncrylamlde s&&on (total acrylamrde con-

tent, %T = 30% w/v, ratio of crosslmkmg agent to
acrylamrde monomer, %C = 2 7% w/w)

Acrylamlde 73 g
BE acrylamrde 28

Dissolve the above and make up to 250 mL m water

This stock solutron IS stable for weeks m brown glass,
at 4C.
(11) Stuck separatzng gel buffer
SDS log
Tns buffer
[2-ammo-2-(hydroxymethyl)- 45 5 g
propane-1,3-droll

Drssolve the above in less than 250 mL of water, adlust

the pH to 8.8 with HCl, and make the volume to 250
mL. This stock solution is stable for months at 4C.
(III) Stock ammomum persulfate.

Ammomum persulfate 10 g

Dissolve in 10 mL of water. This stock solution is stable

for weeks m brown glass at 4C.
(IV) Stock stackmg gel buffer.

SDS log
Trrs buffer 15 1 g

Dissolve the above m less than 250 mL of water, adlust

the pH to 6.8 with HCl, and make up to 250 mL. Check
the pH before use. This stock solution is stable for
months at 4C.
(v) Reservozr buffer (0.192M glycme, 0.025M Tris,
0.1% w/v SDS).

Glycme 28 8 g
Trrs buffer 6.0 g
SDS 2.0 g

Dissolve the above and make to 2 L in water. The solu-

tion should be at about pH 8.3 without adjustment.
This solution is readily made fresh each time.
(VI) Stock (double strength) sample solvent:
SDS Gels 45
SDS 0 92 g
P-Mercaptoethanol 2 mL
Glycerol Jog
7 rls buffer 03i3
Bromophenol Blue 2 mL
(0 1% w/v solution in water)

Dissolve the above in less than 20 mL of water, adlust

the pH to 6.8 with HCl, and make to 20 mL. Check the
pH before use Exposed to oxygen m the air, the
reducing power of the P-mercaptoethanol wanes with
time. Periodically (after a few weeks) add extra agent or
renew the solution. This stock solution 1s stable for
weeks at 4C.
(~122)Protem stain.

Coomassle Brilliant Blue R250 0 25 g

Methanol 125 mL
Glacial acetic acid 25 mL
Water 100 mL

Dissolve the Coomassle dye in the methanol compo-

nent first, then add the acid and water. If dissolved m a
different order, the dyes staining behavior may differ.
The stain 1s best used when freshly made. For best re-
sults do not reuse the stain-its efficacy declines with
use. If this dye 1s not available, use the equivalent dye
PAGE blue 83.
(VW) Destamng solutzon.

Methanol 100 mL
Glacial acetic acid 100 mL
Water 800 mL

Mix thoroughly. Use when freshly made.

Method
1 Thoroughly clean and dry the glass plates and three
spacers, then assemble them as shown in Fig 1, with
the spacers set l-2 mm in from the edges of the glass
46 Smith

plates. Hold the construction together with bulldog

clips. White petroleum Jelly (melted in a boilmg water
bath) IS then applied around the edges of the spacers to
hold them in place and seal the chamber. Clamp the
chamber in an upright, level position
2. A sufficient volume of separating gel mixture (say 30
mL for a chamber of about 14 X 14 X 0.1 cm) is pre-
pared as follows. Mix the following:

Stock acrylamlde solution 15 mL

Distilled water 75mL

Degas on a water pump, and then add.

Stock separating gel buffer 7.5 ml

Stock ammonmm persulfate sol&on 45 FL
N, N, N, N-tetramethyl-
ethylenediamme (TEMED) 15 PL

Mix gently and use immediately (because polymeriza-

tion starts when the TEMED is added). The degassmg
stage removes oxygen, which inhibits polymerization
by virtue of mopping up free radicals, and also discour-
ages bubble formation when pouring the gel.
3. Carefully pipet or pour the freshly mixed solution mto
the chamber without generating air bubbles. Pour to a
level about 1 cm below where the bottom of the well-
forming comb will come when it IS m position. Care-
fully overlayer the acrylamlde solution with butan-2-01
without mixing (to eliminate oxygen and generate a flat
top to the gel). Leave the mixture until it is set (0.5-1.5
l-4.
4. Prepare stacking gel (5 mL) as follows. MIX the
following*

Stock acrylamlde soiutlon 0 75 mL

Distilled water 3 mL
Degas on a water pump, then add.

Stock stacking gel buffer 125 mL

Stock ammomum persulfate solution 15 /.LL
TEMED 5 FL
SDS Gels 47

Mix gently and use immediately.

Pour off the butan-2-01 from the polymerized
separating gel, wash the gel top with water and then a
little stacking gel mixture, and fill the gap remaining in
the chamber with the stacking gel mixture. Insert the
comb and allow the gel to stand until set (about 0.5-l
h)*
5. When the stacking gel has polymerized, remove the
comb without distorting the shapes of the well. Re-
move the clips holding the plates together, and install
the gel in the apparatus. Fill apparatus with reservoir
buffer. The reservoir buffer can be circulated between
anode and cathode reservoirs, to equalize their pH
values. The buffer can also be cooled (by circulating it
through a coolmg coil m ice), so that heat evolved dur-
ing electrophoresis is dissipated and does not affect the
size or shape of protein zones (or bands) in the gel.
Push out the bottom spacer from the gel and remove
bubbles from both the top and underneath of the gel,
for they could partially insulate the gel and distort elec-
trophoresis. Check the electrical circuit by turning
on the power (dc) briefly, with the cathode at the stack-
mg gel end of the gel (i.e., the top). Use the gel
immediately .
6. While the gel is polymerizmg (or before making the
gel), prepare samples for electrophoresis. A dry sample
may be dissolved directly in single-strength sample sol-
vent (i.e., the stock solution diluted twofold with
water) or dissolved m water and diluted with one
volume of stock double-strength sample solvent. The
concentration of sample m the solution should be such
as to give a sufficient amount of protein in a volume not
greater than the size of the sample well. Some proteins
may react adequately with SDS within a few minutes at
room temperature, but as a general practice, heat
sample solutions in boiling water for 2 min. Cool the
sample solution before loading it. The bromophenol
blue dye indicates when the sample solution is acidic
by turnmg yellow. If this happens, add a little NaOH,
enough to lust turn the color blue.
7. Load the gel. Take up the required volume of sample
solution in a microsyrmge or pipet and carefully inject
48 Smith

it into a sample well through the reservoir buffer. The

amount of sample loaded depends upon the method of
its detectlon (see below). Havmg loaded all samples
without delay, start electrophoresis by turning on
power (dc). On a gel of about 0.5-l mm thickness and
about 14 cm length, an applied voltage of about 150 V
gives a current of about 20 mA or so (falling during
electrophoresis if constant voltage is employed) The
bromophenol blue dye front takes about 3 h to reach
the bottom of the gel. Greater voltage speeds up elec-
trophoresls, but generates more heat in the gel.
8. At the end of electrophoresis (say, when the dye front
reaches the bottom of the gel), protem bands m the gel
may be visualized by staining. Remove the gel from be-
tween the glass plates and immerse it m the protein
stain immediately (although delay of an hour or so is
not noticeably detrimental m a gel of 15%T). The gel is
left there with gentle agitation until the dye has
penetrated the gel (about 1.5 h for 15%T gels of 0.5-l
mm thickness). Dye that is not bound to protein is re-
moved by transferring the gel to destaining solution.
After about 24 h, with gentle agitation and several
changes of destaining agent, the gel background be-
comes colorless and leaves protein bands colored blue,
purple, or red. Coomassle Brilliant Blue R250 and
PAGE blue 83 each visibly stain as little as 0.1-l kg of
protein in a band of about 1 cm width.

Notes
1 The reducing agent in the sample solvent reduces inter-
molecular disulphide bridges and so destroys
quarternary structure and separates subumts, and also
oxidizes intramolecular dlsulflde bonds to ensure
maximal reaction with SDS The glycerol is present to
increase the density of the sample, to aid the loading of
it onto the gel. The bromophenol blue dye also aids
loading of the sample, by making It visible, and mdl-
cates the posltlon of the front of electrophoresis in the
gel. The dye also indicates when the sample solution is
acldlc by turning yellow.
2 The polymerization of acrylamlde and blsacrylamlde IS
SDS Gels 49

mitiated by the addition of TEMED and persulfate. The

persulfate activates the TEMED and leaves It with an
unpaired electron This radical reacts with an
acrylamide monomer to produce a new radical that re-
acts with another monomer, and so on to build up a
polymer. The bis acrylamrde is mcorporated mto poly-
mer chains this way and so forms crosslmks between
them.
3. The gel system described is suitable for electrophoresls
of proteins in the M, range of lO,OOO-100,000. Smaller
proteins move at the front or form diffuse, fast-moving
bands, whereas larger proteins hardly enter the gel, if
at all. Electrophoresis of larger proteins requires gels of
larger pore size, which are made by dilution of the
stock acrylamide solution (reduction of %T) or by ad-
justment of %C (the smallest pore size is at 5%C, what-
ever the %T) The minimum %T is about 3%, useful for
separation of proteins of molecular weights of several
millions. Such low %T gels are extremely weak and
may require strengthening by the mclusion of agarose
to 0.5% w/v. Smaller pore gels, for electrophoresis of
small proteins, are prepared by Increasing %T and ad-
justment of %C. Such adjustment of %T and %C may
be found empirically to improve resolution of closely
migrating species.
A combmation of large and small pore gels, suita-
ble for electrophoresis of mixtures of proteins of wide-
ranging sizes, can be made in a gradient gel, prepared
with use of a gradient-making apparatus when pourmg
the separating gel (see Chapter 7).
4. Since proteins (or rather, their complexes with SDS) are
resolved largely on the basis of differences m their
sizes, electrophoretic mobility in SDS gels may be used
to estimate the molecular weight of a protein by com-
parison with protems of known size [as described in
(I)]. However, it should be remembered that some pro-
teins have anomalous SDS-binding properties, and
hence anomalous mobilities m SDS gels.
5. If necessary, the gel may be stored for 24 h (preferably
in the cold) either as the separating gel only, under a
buffer of stock-separating gel buffer diluted fourfold in
water, or together with the stacking gel with the comb
a C

Fig. 2. Examples of proteins electrophoresed on SDS

polyacrylamide (15%T) gels and stained with Coomassie Bril-
liant Blue R 250 as described in the text. Electrophoresis was
from top to bottom. (a) Good electrophoresis. Sample, left,
15qg loading of histone proteins from chicken erythrocyte nu-
clei. Sample right, a 5-kg loading (total) of molecular weight
marker proteins (obtained from Pharmacia). The M,s are, from
top to bottom: phosphorylase b, 94,000; albumin, 67,000; oval-
bumin, 43,000; carbonic anhydrase, 30,000; trypsin inhibitor,
20,100; ol-lactalbumin, 14,400. (b) Examples of artifacts. Sample,
left, the fastest (bottom) band has distorted as it encountered a
region of high polyacrylamide density (which arose during very
rapid gel polymerization). Sample, right, the effect on protein
overloading of increasing a bands size (the sample proteins are
as in sample, left). Extreme overloading may also cause narrow-
ing of faster-migrating bands, as has happened here to some ex-
tent (cf. fast bands widths with widths of bands in sample, left).
(c) Example of an artifact. Sample, left, the end well effect of
distortion of the sample loaded into the very end well, not seen
in samples in other wells (e.g., sample, right).

50
SDS Gels 51

Table 1
Some Problems That May Arise During the Preparatron and Use of
SDS Gels
Fault Cause Remedy
(1) Farlure or a de- (a) Oxygen is pres- (a) Degas the solu-
creased rate of ent tions
gel polymeriza- (b) Stock solutions (b) Renew the stock
tion (especially solutions
acrylamrde
and persul-
fate) are aged
(11) Formation of a Penetration of Overlayer the gel
strcky top to the gel by solution with
the gel butan-2-01 butan-2-01 with-
out mixing them.
Do not leave
butan-2-01 to
stand on a poly-
merized gel
(14 Poor sample wells
(4 The wells are dls- (a) The stacking gel (a) Remove the comb
torted or bro- resists the re- carefully or use a
ken moval of the gel of lower %T
comb
@I The wells contain (b) The comb fits (b) Replace the comb
a loose webbing loosely with a trghter-
of fitting one
polyacrylamlde
(3 Unsatisfactory
staining
(4 The staining IS (a) The dye is (a) Use a more con-
weak. bound meffr- centrated dye so-
ciently lution, a longer
stainmg time, or
a more sensitive
stain The stain
solution should
contain organic
solvent (e g.,
methanol),
which strips the
SDS from the
protein to which
the dye may
then bmd
(contznued)
52 Smith

Table 1 (confznued)
Fault Cause Remedv
(b) The stammg is (b) The dye pene- (b) Agitate the gel
uneven tration or during stammg
destaining is and destammg
uneven Increase the
stammgidestam-
mg time
(4 Stained bands be- (c) The dye has (c) Restain the gel Re-
come decolor- been removed duce the
ized from the pro- destaining time
tein or use a dye that
stams protems
indelibly, e g ,
Procion Navy
MXRB (see
Chapter 14)
(4 The gel is marked (d) Solid dye 1s (d) Ensure full dissolu-
nonspecifically present m the tion of the dye,
by the dye staining solu- or filter the solu-
tion tion before using
1t

(4 Contammants are (a) The apparatus (4 Cl ean or renew

apparent and/or stock them as required
solutions are
contammated
(b) Nonprotem- (b) Try another stain
aceous mate- that will not
rial u-t the stain the
sample (e.g , contaminants
nucleic acid)
has been
stained
(c) Samples have (c) Do not overfill
cross-contam- sample wells.
mated each Ensure good ad-
other because herence of the
of their over- gel layers to each
loading or other by thor-
their sideways ough washing of
seepage be- the polymerized
tween the gel gel before appli-
layers cation of subse-
quent layers
(confrnued)
 SDS Gels 53

Table 1 (contnzued)
Fault Cause Remedy

(vi) Protem bands are (a) Insufficient elec- (a) Prolong the run
not sufficiently trophoresis
resolved
(b) The separating (b) Alter the %T
gels pore size and/or %C of the
is incorrect separating gel
(vii) There are small (a) The amounts (a) Keep the loadmgs
changes m loaded differ roughly similar
standard pro- greatly m size each time
teins electro-
phoretic
mobilmes from
time to time
(b) The constituents (b) Use one batch of a
of the gel vary chemcial for as
in quality long as possible
from batch to Replace aged
batch or with stock solutions
age and reagents
(viii) Distortion of
bands
(4 Bands have be- (a) Proteins m the (a) Use fresh sample
come smeared sample are m- solvent and/or
or streaked soluble or re- extra SDS and
main aggre- reducing agent
gated m the in it (especially
sample sol- for concentrated
vent sample solutions)
There is msolu- Filter the stock so-
ble matter or a lutions before
bubble m the use and remove
gel that has any bubbles
interfered from the gel mix-
with protein tures
band migra-
tion
The pore size of Ensure that the gel
the gel is m- solutions are
consistent well mixed and
that polymenza-
non is not very
(contznued)
54 Smith

Table 1 (contznued)
Fault Cause Remedy
rapid (to slow It
down, reduce
the amount of
persulfate
added)
04 Protein migration Part of the gel (b) Remove any bub-
has been une- has been msu- bles adhering to
ven (bands are lated the gel before
bent) electrophoresls
Electrxal leakage Ensure that the
side spacers are
in place
Coolmg of the Improve the cool-
gel is uneven mg of the gel, or
(allowing one reduce the heat-
part of the gel mg by reducing
to run more the voltage or
quickly than ionic strength of
another) buffers
The band and/or Repeat the electro-
Its neighbors phoresls, but
are overloaded with smaller
loadings. Leave
gaps (I.e., un-
loaded sample
wells) between
nelghbormg
heavily loaded
samples. If nec-
essary, alter the
system [e.g. see
(vz)] and so the
relative
mobllltles of
bands, so that
they do not m-
terfere with each
other
The sample well Avold using the
used was at end wells
the very end
of the row of

(contwzued)
SDS Gels 55
Table 1 (continued)
Fault Cause Remedy
wells (the
end well ef-
fect)
(4 Bands are not of (c) The sample was (c) Check that the
umform thrck- loaded une- sample well bot-
ness venly toms are straight
and horrzontal
[see (m)]

left in place to prevent drying out

6. Proteins dissolved in sample solvent are stable for
many weeks If kept frozen (at -10C or below), al-
though repeated freezing and thawing causes protein
degradation
7. The result of electrophoresrs m SDS gels ideally has
protein(s) as thin, straight band(s) that are well-
resolved from other bands. This may not always be so,
however. Some faults and then remedies are given m
Table 1. Some examples are shown m Fig. 2.
8. Be wary of the dangers of electric shock and of fire, and
of the neurotoxic acrylamide monomer.

References
1 Deyl, Z (1979) EZectrophoreszs A survey of fechnques and applz-
canons Part A* Techntques Elesevrer, Amsterdam
2 Laemmh, U.K (1970) Cleavage of structural proteins during
the assembly of the head of bacteriophage T4 Nature 227,
680-685
3 Studier, F. W (1973) Analysis of bacteriophage T7 early
RNAs and proteins on slab gels J Mel Bzol 79, 237-248