ANALYTICAL BIOCHEMISTRY 1, 391396 (1969)

Preparation and Properties of Acrylamide Gel

for Use in Electrophoresis

SAMUEL RAYMOND AND YI-JU WANG?

From the Pepper Luboratory, University of Pemxsylvartiia,
Philadelphia, Pennsylvania

Received September 15, 1960

The introduction of a new gel medium for electrophoresis requires some

justification in view of the general use and satisfactory resolutions of the
Smithies (4) starch gel. This report presents reasons for considering
acrylamide as a replacement for starch and describes the preparation and
properties of acrylamide gel.
Acrylamide is completely soluble in buffer solutions and polymerizes in
solution to form a suitable supporting medium for gel electrophoresis (3).
When cross-linked with a small proportion of methylenebisacrylamide,
the gel is completely insoluble and stable against temperature or pH
changes. This gel possesses chemical composition and physical structure
which are entirely different from starch and other gels heretofore used. Its
unique characteristics may result in useful differences in ability to effect
separations of different classes of compounds. In addition, we find that
acrylamide gel is superior to starch in physical properties, is prepared
more easily and more reproducibly, and produces protein patterns with
improved definition. On the other hand, it is easier to recover protein
components from starch gel by the freezing technique, which is not ap-
plicable to acrylamide gel since the latter is unaffected by freezing.
The chemical structure of the gel is a linear polymer of acrylamide,
cross-linked at intervals by means of methylene bridges, viz :
-CH&H(CONH)CH&H(CONH2)-

-CH,CH(COP;TH)CH&H(CONHJ-
This structure, which extends in three dimensions, consists of long hydro-
carbon chains made hydrophilic by the amide groups attached at regular
intervals. Except possibly at the terminations of the chains, there are no
1 This research was supported by a grant from the Institute for Cooperative Re-
search, University of Pennsylvania.
Present address: Mount Holyoke College, South Hadley, Mass.
391
392 RAYMOND AND WAN(;

hydroxyl groups or acidic groups in the molecule. Thus this gel differs
markedly from gels of starch or agar, which consist of long polysaccharide
chains, having numerous hydroxyl and acidic groups. Acrylamide gel also
differs from proteins because of the absence of free phenolic, carboxylic,
sulfhydryl and amino groups.

EXPERIMENTAL

Preparation of the Gel

Dissolve 5.0 gm of acrylamide monomer (Cyanogum-41, American
Cyanamid Co.) in 100 ml of the desired buffer. Add 1.0 ml of a freshly
made 10% solution of dimethylaminopropionitrile (DMAPN, American
Cyanamid Co.) in the same buffer and 1.0 ml of a freshly made 10%
solution of ammonium persulfate. Pour into a suitable mold and allow to
stand 3 hr or until gelled.
The gel mold (Fig. 1) is a rectangular box of Plexiglass, 33 X 10 X 5

- Gel. mold

Mold case End spacer Gelform Partition

l$Yk-2 14.39&y 13s. g?$ & 12&.3&&
CUT-OUT 2 6.12

FIG. 1. Gel mold for preparation of acrylamide gel strips 6.5 x 30 cm. Material:
Plexiglass.

cm. Fitting inside it are removable partitions and spacers to form three
slabs of gel each 3 mm thick. By appropriate rearrangement of the parti-
tions, gel slabs up to 9 mm thick can be prepared. Air is excluded during
the gelling period by filling the mold with solution to a level above the
horizontal part of the gelform.
 ACRYLAMIDE GEL 393

Notes on the Preparation

The commercially available monomer mixture usually contains less

than 50 mg of insoluble material per gram, and is used without repurifica-
tion. The insolubles can be removed by filtration or decantation if desired.
Buffers used were formate, acetate, phosphate, barbiturate, EDTA, Tris?
and borate, and also dilute sulfuric acid, with a pH ranging from 1.0 to
11.0. The gel appeared unaffected by the buffer used. The catalyst solu-
tions must be freshly made. The catalyst furnishes free radicals for initia-
tion of the polymerization reaction but does not enter into the structure
of the gel to any significant extent. When the monomer-catalyst solution
is exposed to air, an upper layer to a depth of 1 cm does not polymerize.
The gel mold must be designed with t,he necessity in mind of protecting
the upper layer of solution.
The formulation given produces a gel which is insoluble in all common
buffers and organic solvents and is unaffected by boiling or freezing. On
dehydration it shrinks to a t,hin transparent film; this change is fully
reversible. The flexibility and mechanical strength are comparable to that
of wet filter paper, except in compression.

Horizontal Electrophoresis Cell

Because of the low resistance and high water content of acrylamide gel,
it is essential to use an apparatus in which the gel is in contact with
water-cooled plates on both upper and lower surfaces. Failure to observe
this requirement will result in serious distortion of the gel cause by local
evaporation and overheating. An apparat.us with vapor space, even if
hermetically sealed and cooled externally so that the vapor space is
saturated with water vapor, does not prevent distillation from the surface
of the gel to the walls of the gel enclosure (2).
The apparatus, Fig. 2, used for horizontal gel electrophoresis consists
of a migration chamber 30 X 22 cm with att,ached electrode compart-
ments, each of about 3 1 capacity. The floor of the chamber on which the
gel slabs rest is cooled by tap water circulating through channels in the
base plate. The cover of the migration chamber, also water-cooled, ex-
tends to cover the electrode compartments and is moveable vertically with
respect to the floor. Thus the space between cover and floor adjusts itself
to the exact thickness of the gel used. Electrical contact between the ends
of the gel slabs and the electrode compartments is made by means of
sponges soaked in buffer. The space beneath the cover is entirely filled
with gel, sponge, or buffer, so that no evaporation can take place any-
where.
394 RAYMOND AND WANG

r--7,

/
WATER- COOLED
TROUGH

Electrophorasis apparatus
iiiiijjj
= SPONGE BRFFLES
FIG. 2. Water-cooled electrophoresis chamber, for completely enclosed gel elec-
trophoresis. Material: Plexiglass. Over-all dimensions 40 X 15 X 25 cm.

Application of Speci,men
It is not necessary to precondition or equilibrate the gel in the appara-
tus before applying the specimen. The filter paper technique (4) is used.
Serum specimens from clotted blood were used without further processing.
The razor blade used was broken off to be 15 mm wide, allowing three
specimens to be placed side by side across each gel strip. Place one set
of specimens 4 cm from the negative end and one set 12 cm from the
negative end. This allows six specimens per strip.
Running Conditions
After application of the specimen, cover the gel surface with a sheet of
plastic (Saran-Wrap) eliminating all air bubbles. Place the water-cooled
 ACRYLAMIDE GEL 395

lid in contact with the surface of the covered gel. With cooling water run-
ning at 20-25OC, use 300 v at about 180 ma. When analyzing hemoglobins,
allow the main hemoglobin component to migrate 5-6 cm from its origin
(about 2% hr) . This is sufficient to give well-defined separations. When
analyzing serum proteins, the addition of a small crystal of bromophenol
blue (which colors the albumin component without affecting the electro-
phoretic mobility), allows the progress of the run to be observed visually.
To avoid excessive trailing in serum protein patterns, the current should
be turned off after 5 min (when all the specimen will have moved off the
wick onto the gel strip) and the wick removed from the gel. After closing
up the slit, restart the run.
Dyeing the Patterns
After complet,ion of the electrophoresis run, write identifying data on
the gel with a toothpick dipped in a 5% protein solution, Immerse the gel
in dye solution (e.g., 1% amidoblack 10B Bayer in a mixed solvent con-
taining methanol-water-acetic acid 5:5: 1 v/v) with agitation for 1 min.
Drain and wash in five changes of the mixed solvent until the background
is clear. Finally wash with water until transparency is rest,ored.
The transparency of acrylamide gel is advantageous. A thickness of 3
mm has been adopted as standard in our laboratory. In this thickness, the
optical clarity of the gel throughout the visible range allows stained
patterns to be measured by transmission densitometry, with certain
advantages over reflection densitometry as proposed by Pert et al. (1).
There is no significant light scattering either from the interior of the gel
or from the surfaces of the gel as molded, so that slicing off the outer
layers as recommended by Smithies (4) contributes nothing to the defini-
tion of the pattern and in fact depreciates it.
Because acrylamide is a synthetic, water-soluble monomer of which
the physical and chemical properties are carefully controlled during
manufacture, gels prepared from it are uniform, homogeneous, and re-
producible. Concentration changes during heating or deaeration arc
avoided by the elimination of these steps in the preparation of the gel.
No more than 5% monomer is needed to form a gel optimum strength and
clarity. With this smaller concentration of gel, rates of migration are
correspondingly increased, as shows in Fig. 3.
The gel as prepared contains a small proportion of catalyst, which
consists of a mixture of ammonium persulfate and a tertiary aminonitrile.
Most of the catalyst reacts with the gel in initiating the polymerization,
but there is a small residue free within the gel structure. Although we
were concerned about a possible effect of the residual catalyst on the
electrophoretic patterns, experiments in which the catalyst was removed
by dialysis failed to demonstrate any qualitative or quantitative effect
396 RAYMOND AND WANG

..
.()oyL
1 <r; \
1%
15
-002 2
T z1
I -Gal concuntra?ion \
0 p-l- I-1
1 3 5 7

FIG. 3. Migration of normal adult hemoglobin in TrisEDTA buffer, pH 9, cow

taking different concentrations of a&amide gel. Temperature 25, field strength
15 v/cm.

attributable to the presence of catalyst residue. In several experiments,

two strips of gel were prepared from the same catalyzed monomer solu-
tion. One was exhaustively dialyzed against the buffer used in preparing
the gel, to remove any free catalyst residue. During the dialysis the gel
strip swelled about 20% in all dimensions but appeared otherwise un-
changed. Samples of hemoglobin and of serum were applied to both
dialyzed and the nondialyzed strips which were run simultaneously in the
electrophoresis chamber, after trimming the dialyzed strips to the original
dimensions in length and width. No effect of dialysis could be seen in the
resulting pat,terns except that migration rates in the dialyzed gel were
slightly greater. This may be attributed to the swelling of the gel during
dialysis, which opens up the pores in the gel structure, allowing more free
space for migration.
SUMMARY
Acrylamide is a water-soluble monomer which dissolves in buffers over
a wide range of pH. On polymerization it forms transparent flexible stable
insoluble gels suitable for gel electrophoresis. The gel must be adequately
protected from evaporation during electrophoresis. Suitable apparatus
for preparing the gel and for carrying out the electrophoresis is described.
REFERENCES
1. PERT, J. H., ENGLE, R. E., Woons, K. R., SLEISENGER, M. H., J. Lab. Clin. Med.
54, 57!&84 (1959).
2. RAYMOND, S., Manual of Electrophoresis. E. C. Apparatus Co., Swarthmore, 1959.
3. RAYMOND, S., AND WEINTRAUB, L., Science 130,711 (1959).
4. SMITHIES, O., Biochem. J. 61. 629-41 (1955).