DISC ELECTROPHORESIS IN POLYACRYLAMIDE GELS:

EXTENSION TO NEW CONDITIONS O F PH AND BUFFER

Donald E. Williams and Ralph A. Reisfeld*
Merck Sharp & Dohme Research Laboratories, Division of Merck & Co., Inc.
Rahway, N . J .

Disc electrophoresis was developed by Ornstein and Davis' for the analytical
separation of protein mixtures. By this method proteins are concentrated into
thin starting zones and then separated by the combined action of electrophoresis
and molecular sieving. Available
however, are limited to a few pH
values and to a few constituents.+ The purpose of this paper is to review the
procedures and principles of disc electrophoresis and to present a strategy that is
useful in extending it to new conditions of pH and constituent with considerably
greater convenience than by using the rather complex calculations from the
theory presented by Ornstein.'

TERMINOLOGY
AND DESCRIPTION
DISCELECTROPHORESIS:
Disc electrophoresis is usually performed in small (e.g., 7 X 0.5 cm.) columns
of polyacrylamide gel consisting of 3 sections (FIGURE1) : ( 1) a large-pore anticonvection gel into which the protein sample is introduced; ( 2 ) a large-pore
spacer gel in which the sample is electrophoretically concentrated; and ( 3 ) a
small pore gel in which the sample is separated electrophoretically and by
molecular sieving. Electrode compartments, electrodes, and a power supply complete the arrangement. The necessary ionic constituents are designated as the
trailing ion, the leading ion, and the buffer counterion.
At the beginning af the run (FIGURE 2a) the trailing ion$ is in the two
electrode compartments; the leading ion is in the sample, spacer, and running
gels; and the sample is in the sample gel. The buffer is in all sections of the
apparatus, and there is a pH discontinuity at the spacer gel/running gel interface.
As the run progresses (FIGURE2 b ) a schlieren boundary is observed to move
down from the top of the sample gel. Analysis5 indicates that this is the leading
iodtrailing ion boundary. Moreover, if the sample is colored, one can observe
it being swept up by this boundary and concentrated in it. As the combined
boundary passes the pH discontinuity at the spacer gel/running gel interface, the
schlieren boundary is seen to break up (FIGURE 2 c ) . The leading ion/ trailing ion
boundary continues on, while the protein zone migrates more slowly and is left
behind. With time the protein zone broadens as it moves and may fade from view
or resolve into a number of boundaries. Staining of the gel at the completion of
the run reveals that this is due to separation of the protein into its various
fractions. The comparison of this separation with those obtained in free-boundary
electrophoresis and ultracentrifugation confirms that disc electrophoresis separates proteins both according to charge and size. The extent of this size separaPresent address: National Institute of Allergy and Infectious Diseases, National Institutes
of Health, Bethesda, Md.
i Other procedures have been communicated by Canal Industrial Corporation, Bethesda, Md.
t The correct term is trailing ion constituent, since the trailing ion must be a weakly ionized
species. Ion constituent denotes the ion-forming species irrespective of whether it is actually
ionized or n o t 3 This term is cumbersome, however, so we shall continue to use the term trailing
ion, understanding it to mean trailing ion constituent.
B This analysis can be done by cutting up the column at this stage.
373

Annals New York Academy of Sciences

3 74

ELECTRODE
VESSEL
I

SAMPLE
GEL
I

POWER
SUPPLY

SPACER
GEL

RUNNING
GEL

FIGURE1. Disc electrophoresis is performed in small columns of polyacrylamide gt

consisting of three sections. Electrode compartments, electrodes, and a power supply complete the arrangement.

Williams & Reisfeld: Disc Electrophoresis

CONCENTRATION

375

SEPARATION

m n m
.:.:
......
...-.

LEADING TRAILING
I ON
ION

0 0

PROTEIN

FIGURE
2. In disc electrophoresis the sample is first electrophoretically concentrated into
a thin zone. The components of the zone are then separated electrophoretically and by
molecular sieving.

tion is shown to depend upon the concentration of monomer used in the polymerization of the running gel."

VOLTAGE
AND pH GRADIENT
DISCONTINUITIES
The pH anil constituent requirements of disc electrophoresis can be understood in terms of voltage and pH gradient disc0ntinuities.t Voltage gradients are
important because the velocity of an ion in an electrical field depends upon the
product of the mobility of the ion and the voltage gradient (volts/cm.) of the
electrical field,
dx/dt = V u
where dx/dt is the velocity, V is the voltage gradient, and u the mobility of the
ion or ion constituent. Indeed an ion with a lesser mobility can move as fast as
one with a higher mobility if the mobility-voltage gradient products are equal.
V j ~=
j dx/dt = VZUZ.
In disc electrophoresis such an equality is automatically created and results
in the concentration of the proteins. One can visualize it in this way. The leading
ion is chosen to have a mobility higher than either the proteins or the trailing ion.
The trailing ion is chosen to have a lower mobility than any of the proteins. At

* For example, in a column made up of a 7%% polyacrylamide gel polymerized on top of

a 15% gel, certain serum components are observed to run in the 7%% gel but to stop at the
7%% /15% interface.
t Omstein' has considered these discontinuities and in addition has presented the subject in
terms of the Kohrausch regulating function.

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Annals New York Academy of Sciences

the moment the voltage is applied the leading ions, moving down from the top
of the sample gel, run away from the trailing ions leaving behind a zone of lower
conductivity and, therefore, an increased voltage gradient (FIGURE 3 ) . In this
voltage gradient the trailing ions are accelerated to keep up with the leading ions.
A steady state is set up whereby the equality of the mobility-voltage gradient
products of the leading and trailing ions is established and maintained. A discontinuity in the voltage gradient is also created at the leading iodtrailing ion
boundary, leaving low gradient before the boundary, high after it. This voltage
gradient discontinuity will migrate down the tube with the leading ion/ trailing
ion boundary, and it will rapidly overtake the proteins in front of the boundary.
This happens because the proteins have smaller mobilities than the leading ions
and, with both ions in the same voltage gradient, the proteins have a lower
velocity. Behind the boundary in the higher voltage gradient the proteins will
have a higher velocity; even higher than the boundary because they have higher
mobilities than the trailing ions, and both are in the same voltage gradient. As a
result the boundary will sweep up the proteins and deposit them in a very thin
zone. Indeed, the voltage gradients will counter the effect of diffusion and most
other disturbances.
Now consider the pH discontinuity that is established at the spacer gel/running
gel interface during the preparation of the column. The trailing ion and this pH
gradient are carefully chosen so that the trailing ion will move more slowly than
the slowest protein in the spacer gel and more rapidly than the fastest protein in
the running gel. This is accomplished by choosing a weakly ionized species for
the trailing constituent. The pH discontinuity is so arranged that the degree of
ionization of the trailing constituent, which is very low in the spacer gel, will

ELECTRODE VESSEL

HIGH VOLTAGE
GRADIENT

SAMPLE GEL

DISCONTINUITY
SPACER GEL
LOW VOLTAGE
GRADIENT

RUNNING GEL

TRAILING ION

LEADING ION

FIGURE
3. The fast-moving leading ion leaves behind a region of high voltage gradient.

Williams & Reisfeld: Disc Electrophoresis

377

increase several times as the leading ion/ trailing ion constituent boundary passes
into the running gel. Because of this increase in the mobility of the trailing ion
constituent, the mobility will be more nearly equal to that of the leading ion. This
in turn will alter the voltage gradient, which moves with the boundary. In this
new voltage gradient the proteins will move more slowly than the boundary, and
the boundary will leave the proteins behind. The proteins in a thin starting zone
and in a region of relatively constant pH and voltage gradient will be subjected
to ordinary zone electrophoresis.
To summarize the requirements for disc electrophoresis:
(a) The leading ion must have a mobility that is higher than that of any other
component and is conveniently independent of the pH. It must have the
same sign of charge as the sample at the different pH values in the column.
(b) The trailing ion constituent must be an amino acid, a weak acid, or a weak
base. In its ionic form it must have the same sign of charge as the leading
ion. It must have a low net charge in the sample and spacer gels and higher
net charge in the running gel.
( c ) The pH discontinuity between the spacer and running gels must properly
program the mobility of the trailing ion constituent.
( d ) The buffer must supply counter ions for electrical neutrality and must buffer
the column. T o do this it must be a weak acid or weak base with a sign of
ionic charge opposite to that of the leading ion. The pH of maximum
buffering should be close to the pH of the running gel.
A practical approach to the design of disc electrophoresis systems is based on
these requirements. Such an approach is stated and discussed in the following
section.

DESIGN
OF DISCELECTROPHORESIS
SYSTEMS:
A GENERAL
FORMULATION
Anionic Systems
1. Choose a pH f o r the running gel. Experimental determinations and theoretical calculations1 of the actual running pH show it to be commonly approximately one-half pH unit higher than the initial pH of the running gel. With this
in mind, considerations of maximum resolution, chemical stability, biological
stability, and sample solubility will dictate this choice of pH. Moreover, all the
proteins must have the same sign of charge at the pH chosen.
2 . Use chloride ion as the leading ion. The chloride ion is an ideal choice. It
has a high electrophoretic mobility that is independent of pH. As a component
of most biological systems, it has few damaging effects on proteins.
3 . Choose as the trailing ion a weak acid or amino acid with a pKa up to one
pH unit higher than that o f the running gel. This insures that about half the
molecules of the weak acid will have a net charge at the actual running pH and
that the ion constituent mobility will be higher at this pH than any of the protein
mobilities.
4. Choose as the pH o f the sample and spacer gels a value 2 to 3 pH units
1pss than the p K a of the trailing ion. Under these conditions 1 to 0.1 per cent of
the molecules of the weak acid will have a net charge, and the ion constituent
mobility will be very low. If this mobility should prove to be higher than that of
the slowest moving protein, go back to step 1, choose a higher pH for the running
gel, and proceed again with steps 2 , 3 , etc.
5 . Choose as the bufler a base with a pK, u p to one pH unit less than that of
running gel. This allows the desired high buffering capacity in the running gel.

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Annals New York Academy of Sciences

It does, however, leave the sample and spacer gels with little buffering, so care
must be taken that the p H is not changed by contamination. In some cases the
pH of the sample must be adjusted to that of the sample gel solution before
mixing. Depending on the pKs of the buffer, it may be necessary to change the
sample pH slightly. It is best to keep the sample p H within 1% p H units of the
buffer pK,. Ornstein" has found that the use of a buffer for the leading ion in
the sample and spacer gels will solve the buffering problem. To avoid confusion
all instructions are expressed in terms of pK,. When tables of pKb's are used, the
pK, is found by subtracting the pKb from 14.
6. Make the p H in the electrode vessel equal to the ply, of the buffer. The p H
in the electrode vessel is not critical but making it up this way minimizes the pH
changes caused by the products of electrolysis.
7. The stock solutions are made u p in standard fashion,'J2except as indicated
below .
Stock A
1N HCl
48 ml.
Enough buffer to obtain pH of running gel.
0.46 ml. TEMED'
qs. 100 ml.
* N,N,N,N,Tetramethylethylenediamine.

Stock B
48 ml.
Enough buffer to obtain pH of sample gel.
0.46 ml. TEMED*
qs. 100 ml.

1N HCI

The additions of the buffer are made with the aid of a pH meter. This method is
faster and usually more accurate than calculations using the Henderson-Hasselbalch equation. (Once the quantities are established by this method they can, of
course, be added directly without using the pH meter each time.)
Cationic Systems
Cationic Systems are set up in the same way as above except for the following
changes where the numbers refer to the above paragraphs: ( 1 ) The actual
running p H will be ?/z pH unit less than the initial pH of the running gel; (2) Use
the potassium ion in place of chloride; ( 3 ) The trailing ion is a weak base or
amino acid with pK, up to one pH unit less than the p H of the running gel;
(4) The p H of the sample and spacer gels is between 2 and 3 p H units more
than the pK, of the trailing ion; ( 5 ) The pK, of the buffer is up to one pH unit
higher than that of the running gel; ( 7 ) I N KOH is substituted for I N HCI.

DESIGN
OF DISCELECTROPHORESIS
SYSTEMS-EXAMPLES
To illustrate the general approach given above, consider the anionic system
of Ornstein and Davis' and the cationic system of Reisfeld, Lewis, and Williams.2
They can be used to compare our strategy to practice (TABLE 1) .
Ornstein and Davis use a p H of 8.9 in the running gel or a running p H of
about 9.4. At this p H nearly all proteins are soluble and negatively charged.
Many protein mixtures are well resolved and are chemically and biologically
stable. This pH is good for a large number of applications. In this anionic system the chloride ion is used as the leading ion. Glycine with a pK, of 9.8 fits the
requirement that it be between 8.9 and 9.9. At 6.7 the pH of the sample gel is
slightly lower than the ideal range of 6.8 to 7.8. A pH of 6.7 is a reasonable
value, however, since it is only 1.4 p H units less than the pK, of the buffer.
2-Arnino-2-h~drox~methyl-1,3-propanediol
(TRIS) is a good choice for the
* Personal communication. Ornstein substitutes phosphate for the chloride in his standard
procedure.'

Williams & Reisfeld: Disc Electrophoresis

379

TABLE
1
EXISTING
Disc ELECTROPHORESIS
SYSTEMS:
COMPARISON
WITH THISSTRATEGY
(a) Anionic system with running gel
pH of 8.9
pK. of trailing ion
pH of sample gel
pK, of buffer
pH of electrode buffer

This strategy
8.9-9.9
6.8-7.8
1.9-8.9
8.1

Ornstein and Davis

9.8
6.7
8.1
8.3

This strategy
3.3-4.3
5.6-6.6
4.3-5.3
4.8

Reisfeld, Lewis,
and Williams
3.6
6.8
4.8
4.5

(b) Cationic system with running gel

pH of 4.3

pK. of trailing ion

pH of sample gel
pK. of buffer
pH of electrode buffer

buffer, with a pK, of 8.1 well within the required range of 7.9 to 8.9. The pH of
the electrode buffer at 8.3 is quite close to the suggested value of 8.1. It is apparent that a system derived from our general approach would be nearly identical
with that of Ornstein and Davis.
The cationic system of Reisfeld, Lewis, and Williams (TABLE l b ) uses a p H of
4.3 for the running gel, which lowers to about 3.8 under running conditions.
This is a good pH for the resolution of proteins that are cationic at this pH.
Solubility and stability are generally satisfactory. Potassium ion is used as the
leading ion in agreement with our strategy. The trailing ion, p-alanine, with a
pK, of 3.6, is in the proper range (3.3-4.3). The pH of the sample gel, however,
is too high. It is 0.2 units higher than the theoretical range but, much more importantly, it differs by 2.0 pH units from the buffer pK,. The sample and spacer
gels would be better buffered if the sample gel pH were 6.3. The use of acetic acid
(pK,, 4.8) as-the buffer is in line with our strategy (4.3-5.3), and the pH of the
electrode vessel (4.5) is close to the suggested 4.8.With the exception of the
sample gel pH, this system is very close to the theoretical one.
Not every pH or every constituent that one might wish to employ can be used
in the design of practical schemes for disc electrophoresis. The difficulty is not
one of theory but one of practice. Weak acids or bases with the proper pK,
values must be found. Most often very few are available in the interesting ranges.
Even if weak acids or bases with the proper pK,s are found, they must have a
water solubility of at least 0.1M; they must be available; they must not be too
toxic or explosive to use; and they must not damage the protein.
The design of a new anionic system will illustrate some of these difficulties.
This system has been tested and does work, but it has limitations as will become
apparent. A running gel pH of 7.5 or a running pH of 8.0 was chosen. This is a
pH of maximum resolution for proteins that are ionized to different degrees at
this pH but that tend to have nearly the same charges at higher pH values. Moreover, stabilities of proteins are better than at the higher pH values, especially
for many enzymes. While a weak acid with a pK, between 7.5 and 8.5 was sought
for the trailing ion, diethyl barbituric acid (Verona]) with a pK, of 7.4 was
chosen (TABLE 2 ) . This pK, is perhaps somewhat low, but it is the best we could

Running gel pH of 7.5

This strategy

New system

pK, of trailing ion

7.5-8.5

7.4

pH of sample gel

4.4-5.4

5.5

pK. of buffer
pH of electrode buffer

6.5-7.5

8.1

8.1

7.0

find. Our theories suggest a sample gel pH between 4.4 and 5.4, but lack of a
really satisfactory buffer made it necessary to use pH 5.5. This buffer was to have
a pK, between 6.5 and 7.5, but after many possibilities were eliminated because
of insolubility, toxicity, unavailability, and even explosiveness, TRIS with a pK,
of 8.1 was finally chosen. With a pK, such as this the sample gel, even at pH
5.5, is very poorly buffered. According to our instructions the electrode vessel
buffer must have a pH of 8.1 equal to the buffer pK,, but the theory is not intended for a pK, so far from the ideal. A pH of 7.0 was selected for the
electrode buffer in order not to disturb too greatly the pH at the top and bottom
of the column.
The details of the system are as follows:
Stock A: 48 ml.
1N HC1
Stock B: 48 ml.
1N HCI
6.85 gm. TRIS
4.95 gm. TRIS
0.46 ml. TEMED
0.46 ml. TEMED
qs. 100 ml.
qs. 100 ml.
pH 7.5
pH 5.5
Electrode Buffer: 5.52 gm. Diethylbarbituric acid
1.0 gm. TRIS
qs. 1 liter
pH 7.0
The other stock solutions are the same as those of Ornstein and Davis.
For practical applications we recommend that Stock B be made up as follows:
39 ml. 1M H3P04
4.95 gm. TRIS
0.46 ml. TEMED
qs. 100 ml.
pH 5.5
The substitution of phosphoric acid for HC1 greatly improves the buffering.
Some may be tempted to conclude from the discussion of the last system that
the restrictions given in the foregoing sections are too stringent or unnecessary.
It may seem that almost any combination of pH and constituent will work. This
is not so. In the pH 7.5 system the pH and voltage gradients are in the correct
ranges. The difficulty is the lack of adequate buffering, which can be overcome
to some extent by careful preparation of the column and samples. If, however,
the pH or voltage gradients are not in the correct ranges the sample will not be
concentrated. In one experiment in which the difference between the pK, of the
trailing ion and the sample pH was insufficient, no permanent trailing ion/leading
ion boundary was formed, and the sample was not resolved in the running gel.

Williams & Reisfeld: Disc Electrophoresis

38 1

REFERENCES
1. ORNSTEIN,
L. & B. J. DAVIS.Disc electrophoresis. Unpublished work. Preprinted by Distillation Products Industries, Div. Eastman Kodak co. Rochester, N. Y.
2. REISFELD,R. A,, U. J. LEWIS& D. E. WILLIAMS.1962. Disc electrophoresis of basic
proteins and peptides on polyacrylamide gels. Nature 195(4838) : 281-283.
3. MACINNES,
D. A. 1961. The Principles of Electrochemistry : 60. Dover Publications, Inc.,
New York, N. Y.