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Speeding up SDS-PAGE: theory and experiment

Article in Electrophoresis · April 2023

DOI: 10.1002/elps.202300011

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DOI: 10.1002/elps.202300011

RESEARCH ARTICLE

Speeding up SDS–PAGE: Theory and experiment

Maria K. Koshkina1 Mikhail D. Shelomov1 Anastasia A. Pometun1,2
Svyatoslav S. Savin1 Vladimir I. Tishkov1,2 Denis L. Atroshenko1,2

1 Department of Chemical Enzymology,

Faculty of Chemistry, Lomonosov
Moscow State University, Moscow,
Russian Federation
2 Bach Institute of Biochemistry, Federal
Research Centre “Fundamentals of
Biotechnology” of the Russian Academy
of Sciences, Moscow, Russian Federation
Correspondence
Denis L. Atroshenko, Senior Research
Scientist at the Department of Chemical
Enzymology, Faculty of Chemistry,
Lomonosov Moscow State University,
Leninskie Gory 1-3, 119991 Moscow,
Russian Federation.
Email: atrdenis@gmail.com
Abstract
In order to accelerate Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE), here we propose an optimized version of the technique enabled by
experimental tuning reinforced by theoretical description. In the resulting sys-
tem, the gel buffer was diluted twofold and supplemented with glycine at a low
concentration, whereas a higher voltage was applied. This approach reduced run-
time from 90 to 18 min. It is important to emphasize that, despite the high voltage
applied to the gel, the resolution of the bands did not decrease compared to the
original Laemmli method. The proposed acceleration approach can be used in
other variants of SDS–PAGE.
KEYWORDS
Laemmli electrophoresis, protein electrophoresis acceleration, SDS–PAGE

Color online: See the article online to

view Figures 1, 2, 4 and 5 in color.

1 INTRODUCTION developed by Laemmli in 1970 [4]. The system was later

Sodium dodecyl sulfate–polyacrylamide gel electrophore-
sis (SDS–PAGE) is one of the most commonly used protein
analysis methods in a biochemical laboratory. The devel-
opment of the method started shortly after the discovery
of the electrophoresis principle as the migration of charged
particles in an electric field. Applied to a porous matrix, it
allowed us to separate particles or molecules. Earlier vari-
ations of the technique used starch gel [1, 2], but later it
was substituted with polyacrylamide as a more convenient
medium. In 1964, Ornstein and Davis proposed the prin-
ciples of discontinuous electrophoresis [3], which allowed
us to enhance the separation. Modern SDS–PAGE was
Abbreviations: APS, ammonium persulfate; TGB, Tris-glycine buffer.
Maria K. Koshkina and Denis L. Atroshenko contributed equally to this
manuscript.
modified for autoradiographic analysis of labeled proteins
by Douglas et al. [5]. In addition, several derivative meth-
ods, such as Tricine-SDS–PAGE [6], Blue-native PAGE [7],
have been developed. A more recent study suggested a way
of separating peptides in a Laemmli system using a lower
concentration of SDS [8]. In addition, it is important to
mention that Western [9] and Eastern [10] blot techniques
are also based on SDS–PAGE as their first step.
However, the Laemmli SDS–PAGE technique, which
remains the most widely used, has not undergone sig-
nificant improvements since its development 50 years
ago. Commercially available rapid precast SDS–PAGE gels
and kits are significantly more expensive than hand-cast
gels. Moreover, their undisclosed composition cannot be
modified at the discretion of the researcher.
Depending on the length of the gel and the applied volt-
age, SDS–PAGE can take from 1.5 to 5 h. Considering the

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© 2023 The Authors. Electrophoresis published by Wiley-VCH GmbH.

Electrophoresis 2023;1–10. www.electrophoresis-journal.com 1

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2 KOSHKINA et al.

F I G U R E 1 Component distribution in the gel. The cathode is above, and the anode is below. Green represents Tris+ /Cl− system,
yellow—Tris+ /Gly− , red—the proteins. TGB stands for Tris-glycine buffer.

time required for gel preparation, staining and destaining,
an entire process is labor intensive and time-consuming.
Therefore, new approaches to accelerating SDS–PAGE are
required.
In the current work, we report on modifications to the
Laemmli SDS–PAGE system that reduce the typical run-
time down to 18 min with no sacrificing resolution or
alterations in separation properties.
2 THEORETICAL ASPECTS
In an effort to get insights to the processes that take place
in the gel during electrophoresis, we described the system
as a series of variable resistance resistors. This approach
allowed us to draw certain theoretical conclusions pre-
sented later.
2.1 Change of the gel buffer
composition over time
The most commonly used SDS–PAGE variant described
by Laemmli uses a gel consisting of two parts. The upper
part has a pH of 6.8, whereas the lower part has a pH of
8.8, as presented in Figure 1. The Tris buffer is a part of
both the gel and the running buffer, a mobile phase that
moves across a stationary matrix. However, although in
the gel buffer, a counterion is chloride Cl− , in the running
buffer, it is glycinate Gly− . During the electrophoresis,
the boundary between Cl− and Gly− ions moves toward
the anode. As Gly− has lower mobility in the electric
field, the boundary is not disrupted. The proteins con-
centrate between the Cl− and Gly− fronts. This way
discontinuous electrophoresis narrows the protein bands
and thus enhances resolution. After the proteins enter
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KOSHKINA et al. 3

TA B L E 1 List of designations.
Designation Description
US Total voltage
RA Anode buffer resistance
RC Cathode buffer resistance
RAC RA + RC
RG Gel resistance before the Cl− /Gly− boundary
F I G U R E 2 Four resistors scheme, where RA stands for anode RGstart Initial gel resistance
buffer resistance, RC —cathode buffer resistance, RG —resistance
RP Gel resistance after the Cl− /Gly− boundary
before the Cl− /Gly− boundary, RP —resistance after the Cl− /Gly−
RPend Final gel resistance
boundary, RGstart —initial resistance, and RPend —terminal resistance.
The purple dotted line indicates the boundary. RS Total resistance
RSstart Total initial resistance RAC + RGstart
L Cl− /Gly− boundary position
the lower gel, they separate according to their molecular lG Gel length before the Cl− /Gly− boundary
masses. As suggested by Ornstein [3], the Cl− /Gly− ratio lend Total gel length
at the boundary and the lower Tris/upper Tris ratio are
φ RPend /RGstart
determined by Equations (1) and (2), respectively. Based
V The velocity of the Cl− /Gly− boundary
on these equations, concentrations of the components in
T The time from the beginning of the
certain parts of the gel during the Laemmli electrophoresis
electrophoresis
were calculated, as shown in Figure 1:
tend The total time
𝑚Gly− 𝑧Cl− (𝑚Cl− − 𝑚Tris+ ) K The ratio between the Cl− /Gly− boundary
𝐶Gly = 𝐶Cl− ( ) (1) velocity and the electric field strength in the
𝑚Cl− 𝑧Gly− 𝑚Gly− − 𝑚Tris+ lower part of the gel

𝐶Tris_upper = 𝐶Tris_lower
( ) difference between them will be k constant value. We will
𝑚Tris+ 𝑧Cl− 𝑚Cl− − 𝑚Gly− now consider the lower part of the gel, so the electrophore-
−𝐶Cl− ( ) (2)
𝑚Cl− 𝑧Tris+ 𝑚Gly− − 𝑚Tris+ sis speed will be determined by the strength of the electric
field in it.
Generally, these equations can be derived from the
Kohlrausch function (KRF) and solution electroneutrality
law, as shown in the following equation: 2.2.2 Time and heat

∑
𝑁
|𝑧i |𝑐i (𝑥, 𝑡) This model allows us to derive the following equations
KRF (𝑥) = (3) (for derivation see Section 2.3), which determine the
𝑖=1
𝑢i
dependence of time and heat on other variables in the
system.
2.2 Four resistors scheme
2 𝜑+1
𝑙end 𝑅Gstart + 𝑅A + 𝑅C
2.2.1 Scheme description 𝑡end = 2
(4)
𝑘𝑈S 𝑅Gstart
To understand the processes leading to the desired elec-
trophoresis acceleration, the system can be presented as
( (√ )
a series of resistors, as depicted in Figure 2 and listed in 𝑈𝑆2 2
Table 1. For simplicity, the lower and the upper parts of the 𝑄end = 2 𝑅Sstart + 𝛼𝑡 − 𝑅Sstart
𝛼
gel are considered here as one resistor RG . ( ))
During the electrophoresis, the gel buffer composition 𝛼
− (𝑅A + 𝑅C ) ln 1 + 𝑡 (5)
changes. As a result, the resistance shifts from RGstart to 2
𝑅Sstart
RPend . The gel resistance is defined by the Cl− /Gly− bound-
ary position. To determine the electrophoresis velocity, 2
2(𝜑−1)𝑅Gstart 𝑘𝑈S
where 𝛼 =
both the upper and lower part of the gel could be used. The 2
𝑙end
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4 KOSHKINA et al.

As equations for both time and heat are derived, it is
possible to strictly define ways of the electrophoresis accel-
eration, see Figure S1. The tend should be minimal, whereas
the Qend should at least not increase significantly.
Total time is defined by the Equation (4). This relation
shows that the electrophoresis time can be shortened by
four main ways:
∙ Increase US
∙ Increase RGstart
∙ Decrease RAC
∙ Decrease φ
Let us briefly consider each of them.
Taking into account Equations (1) and (2), it is clear that
𝛼1 𝑐𝑜𝑛𝑠𝑡1 𝐶Tris_lower + 𝑐𝑜𝑛𝑠𝑡2 𝐶Cl
𝜑≈ ( ) (7)
𝛼2 𝑐𝑜𝑛𝑠𝑡3 𝐶Tris_lower + 𝑐𝑜𝑛𝑠𝑡4 𝐶Cl
where const1 , . . . , const4 are constants composed of ion
mobilities and charges. Thus, simple dilution of the gel
buffer, which leads to a growth of initial gel resistance,
does not influence φ. It depends on buffer composition
and pH. It causes further complications because pH and
the buffer ions mobilities in Laemmli–Ornstein system are
chosen to match protein mobilities and perform stacking
and separating.
Consequently, dilution with a corresponding voltage rise
would be the best way of acceleration.

2.2.3 Increasing US
2.3 Derivations
The rise of voltage is the easiest and technically feasible
way. However, as Equation (5) states, total heat Q, emit- Equation (4):
ted across the gel, rises as a square of the voltage. Thus,
it becomes the main limitation to the voltage increase. 𝑅G
𝑈G = 𝑈S
Excessive heat lowers resolution and widens the bands. 𝑅S
( )
𝑙
𝑅Gstart 1 −
𝑙end
2.2.4 Increasing RGstart = 𝑈S (
𝑙
)
𝑙
𝑅A + 𝑅C + 𝑅Gstart 1 − + 𝜑𝑅Gstart
𝑙end 𝑙end
Consequently, the voltage rise should be followed by a ( )
𝑙
corresponding RGstart (initial resistance) rise. It should be 𝑅Gstart 1 −
𝑙end
noted that the resistance rise not only reduces heating but = 𝑈S ( ) (8)
𝑙
also shortens the time (4). In practice, the RGstart can be 𝑅A + 𝑅C + 𝑅Gstart 1 + (𝜑 − 1)
𝑙end
increased by simply diluting the gel buffer.
( )
𝑙
𝑙G = 𝑙end − 𝑙 = 𝑙end 1− (9)
2.2.5 Decreasing RAC 𝑙end

Another option is decreasing the RAC , which is the sum Velocity by definition is as follows:
resistance of the anode and cathode buffer. But with its
𝑑𝑙
decrease, Q significantly rises, making this way disadvan- 𝑣 = (10)
𝑑𝑡
tageous.
Taking in the account Equations (8) and (9),
2.2.6 Decreasing φ 𝑈 𝑅
𝑑𝑙
𝑣= = 𝑘 G = 𝑘𝑈S Gstart
𝑑𝑡 𝑙G 𝑙end
A few words should be said on the nature of the φ
parameter, as it is less obvious than the previous three. 1
× ( ) (11)
By definition, φ equals to the ratio between the final and 𝑅A + 𝑅C + 𝑅Gstart 1 + (𝜑 − 1)
𝑙
𝑙end
initial resistance of the gel. The resistance of the solution
is inversely proportional to the mobility of the ions multi-
plied by their concentration and the degree of dissociation. ( ( ))
𝑙
Then 𝑅A + 𝑅C + 𝑅Gstart 1 + (𝜑 − 1) 𝑑𝑙
𝑙end
𝑅Pend 𝛼1 𝐶Tris_lower 𝑚Tris + 𝐶Cl 𝑚Cl 𝑘𝑈S 𝑅Gstart
𝜑 = ≈ (6) = 𝑑𝑡 (12)
𝑅Gstart 𝛼2 𝐶Tris_upper 𝑚Tris + 𝛼2 𝐶Gly 𝑚Gly 𝑙end
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KOSHKINA et al. 5

By solving the differential equation, we get: TA B L E 2 Gel casting.

Solution Lower gel Upper gel
(𝜑 − 1) 𝑅Gstart 2 Volume, mL
𝑙 + (𝑅A + 𝑅C + 𝑅Gstart ) 𝑙
2𝑙end AA 2 0.5
𝑘𝑈S 𝑅Gstart Ugb – 0.19
= 𝑡 + const (13)
𝑙end Lgb 0.94 –
Distilled water 1.9 2.3
If t = 0 and l = 0, then const = 0, thus APS 0.05 0.03

(𝜑 − 1) 𝑅Gstart 2 SDS 0.05 0.03

𝑙 + (𝑅A + 𝑅C + 𝑅Gstart ) 𝑙 TEMED 0.004 0.003
2𝑙end
Abbreviations: AA, acrylamide; APS, ammonium persulfate; Lgb, lower gel
𝑘𝑈S 𝑅Gstart buffer; SDS, sodium dodecyl sulfate; Ugb, upper gel buffer.
= 𝑡 (14)
𝑙end

2 𝜑+1
𝑙end ( 𝑅Gstart + 𝑅A + 𝑅C ) Integration gives us the final equation for total heat:
2
𝑡end = 𝑡 (𝑙 = 𝑙end ) = (15)
𝑘𝑈S 𝑅Gstart
( (√ )
𝑈𝑆2 2
Equation (5): Q = 2 𝑅Sstart + 𝛼𝑡 − 𝑅Sstart
𝛼
Thermal power emitted across the gel: ( ))
𝛼
𝑅G + 𝑅P − (𝑅A + 𝑅C ) ln 1 + 2
𝑡 (22)
𝑃 = 𝑈G 𝐼G + 𝑈P 𝐼P = 𝑈S2 (16) 𝑅Sstart
𝑅S2

Dependence of thermal power from time is nonlinear; 3 MATERIALS AND METHODS

thus, total heat can be found by integrating:

𝑅G + 𝑅P
3.1 Gel preparation
𝑑𝑄 = 𝑈S2 𝑑𝑡 (17)
𝑅S2
To prepare the gels, several stock solutions were used. The
( ) upper gel buffer (Ugb) contained 1 M Tris HCl, pH 6.8.
𝑙
𝑅G + 𝑅P = 𝑅Gstart 1 + (𝜑 − 1) (18) The lower gel buffer (Lgb) contained 0.8 M C(Tris), 0.2 M
𝑙end
C(Glycine), ≈0.15 M C(HCl) (pH was adjusted to 8.8 with a
Extracting l from Equation 14, pH meter by adding concentrated HCl to the solution).

√ 2
2 2(𝜑−1)𝑅Gstart 𝑘𝑈S
− (𝑅A + 𝑅C + 𝑅Gstart ) + (𝑅A + 𝑅C + 𝑅Gstart ) + 2 𝑡
𝑙end
𝑙 = 𝑙end (19)
(𝜑 − 1) 𝑅Gstart

From Equations (17) to (19), Acrylamide-bisacrylamide solution was prepared in dis-

√ tilled water: T = 30%, C = 3% (mass concentration of
2
2(𝜑−1)𝑅Gstart 𝑘𝑈S
2
− (𝑅A + 𝑅C ) + 𝑅Sstart + 𝑡 acrylamide = 29% and bisacrylamide = 1%).
2
𝑙end Solutions of SDS (SDS) and ammonium persulfate were
𝑑𝑄 = 𝑈S2 2
𝑑𝑡
2 2(𝜑−1)𝑅Gstart 𝑘𝑈S prepared in water with a mass concentration of 10%.
𝑅Sstart + 2 𝑡
𝑙end Gel was cast according to Table 2; volumes given are
(20) quantified for a Mini-PROTEAN gel (8.3 cm × 7.3 cm
Let × 0.75 mm).
2 First, the lower gel was cast layered with ethanol to pre-
2 (𝜑 − 1) 𝑅Gstart 𝑘𝑈S
𝛼 = (21) vent oxygen exposure and then allowed us to polymerize
2
𝑙end for approximately 15 min. Then the ethanol was removed,

6 KOSHKINA et al.
and the upper gel was cast on top of the lower one. The
polymerization took approximately 15 min.
The resulting gel should have the following compo-
sition: C(Tris) = 65 mM, C(HCl) = 62 mM in the Ugb;
C(Tris) = 150 mM, C(Gly) = 38 mM, and C(HCl) = 31 mM
in the Lgb. This corresponds to a twofold diluted 20%
glycine fast gel. This composition was achieved by con-
ducting a series of tests, in which gels with different
dilutions and glycine concentrations were used. Dilution
was achieved by adding a smaller amount of buffer to the
gel and filling the difference with water. Glycine concen-
tration in the Lgb measured relatively to the concentration
of Tris (C(Tris) = 188 mM, as glycine was added only to
twofold diluted gels). For exact, buffer compositions see
Table S1.
3.2 Sample preparation
To test the described method, six proteins with different
molecular weights (Sigma Kit No. MW-SDS-70L Molec-
ular weight markers: α-lactalbumin [14.2 kDa], trypsin
inhibitor [20.1 kDa], trypsinogen [24 kDa], carbonic anhy-
drase [29 kDa], egg albumin [45 kDa], and bovine albumin
[66 kDa]) and Escherichia coli cell lysate were used. The
samples were incubated in a loading buffer consisting of
50 mM Tris-HCl (pH 6.8), 100 mM β-MeEtOH, 1% SDS,
0.0025% bromophenol blue, and 10% glycerol for 5 min at
95◦ C. Concentration of α-lactalbumin and egg albumin in
the sample was 150 µg/mL, whereas for other proteins, it
was 75 µg/mL. Thus, applying 20 µL of the sample, we got
1.5 and 3.0 µg of corresponding proteins per band. E. coli
lysate was loaded with 300 µg per band.
3.3 Running buffer
Before running the gel and applying the samples, ∼500 mL
of running buffer consisting of Tris-base (25 mM) and
glycine (250 mM) and SDS (0.1%) was added to the
electrophoretic cell.
3.4 Electrophoretic conditions
All gels were run in Bio-Rad Mini-PROTEAN Vertical
Electrophoresis Cell, 4-gel, for 0.75 mm thick hand-cast
gels and with PowerPac Basic Power Supply. For 600 V,
LKB BROMMA 2297 MACRODRIVE 5 Constant Power
Supply was used. The gel was loaded in the electrophoretic
chamber. Initially, a lower voltage was applied to the gel
until the tracking dye and the samples reach the lower
separating gel. Then a higher voltage was applied.
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3.5 Membrane transfer and staining
conditions
Several stock solutions were used to perform the mem-
brane transfer and staining. The transfer buffer contained
25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3.
The Tris-buffered saline with Tween 20 (TBST) buffer
contained 20 mM Tris HCl, pH 7.5, 150 mM NaCl, and
0.1% Tween 20. Ponceau S staining buffer contained 0.2%
(w/v) Ponceau S and 5% glacial acetic acid. For the trans-
fer, a Merck Immobilon PVDF Membrane and a Bio-Rad
Protean III electrophoretic cell were used.
4 RESULTS AND DISCUSSION
Conclusions drawn theoretically in the second paragraph
were examined on the most common Laemmli SDS–PAGE
system.
4.1 RGstart calculation
Gels shown in Figure 3 were run under low voltage
with increased amount of Tris-glycine buffer (TGB) run-
ning buffer to minimize excessive heat emission. This
allowed us to calculate RGstart , (as presented in Figure 4B).
RGstart was found from linearization. If we measure volt-
age and current at the initial moment of time, by Ohm’s
law, we obtain sum resistance RS . As RGstart increases
proportionally to dilution D we get:
𝑅S = 𝑅AC + 𝐷𝑅Gstart (23)
Considering that RAC is constant RGstart can easily be
obtained. RGstart = 1.1 ± 0.1 kOhm, RAC = 3.7 ± 0.3 kOhm.
4.2 tend as a function of RGstart
Equation (4) predicts that the dependence has the follow-
ing form:
𝐵
𝑡end = 𝐴 + (24)
𝑅Gstart
where A and B are constant.
As Figure 4A shows, the function indeed resembles a
hyperbola. Thus, the adequacy of the mathematical model
was confirmed experimentally. But more importantly than
that, the function is decreasing, which means that total
time becomes shorter with the resistance increase caused
by dilution.
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KOSHKINA et al. 7

F I G U R E 3 Gels conducted at 100 V, Laemmli buffer diluted, respectively, one-to-fourfold. Adobe Photoshop was used to uniformly
adjust the contrast of an entire image.

F I G U R E 4 (A) Dependence of total time tend on initial gel resistance RGstart . The resistance was calculated from gels presented in
Figure 3, see Section 4.1 RGstart calculation. (B) Dependence of total resistance RS on dilution of the gel buffer.

4.3 Dilution and voltage rise
As predicted by theory, the buffer dilution resulted in
an acceleration of separation and a decrease of current.
Hence, we increased voltage for further acceleration. For
that purpose, twofold dilution was chosen, as it is closer
to the original system. As Figure 5A(left) shows, under
higher voltage an undesirable effect was observed—the
smallest protein was absent on the gel. The reason behind
this is yet unknown. Moreover, dilution led to a resolution
decrease as demonstrated in Figure 5B. The most likely
reason for this could be a drop in the gel’s buffer capacity.
To offset for these drawbacks, several additives to the gel
buffer were examined. One of them—glycine—showed an
interesting property—it returned the smallest protein to
the gel and generally reduced the average Rf , as shown in
Figures 5A(right),C, respectively. Besides, the addition of
glycine also enhanced the resolution, as seen in Figure 5D.
Different concentrations of glycine were tested. Concen-
tration of 38 mM (20% of Tris concentration in original
buffer) was found to be sufficient to maintain the resolu-
tion under the voltage rise. Different voltages were tried
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8 KOSHKINA et al.

F I G U R E 5 (A) Comparison of gels conducted at 150 V/300 V, Laemmli buffer twice diluted with (right) and without (left) 20% glycine
added. The number of percents above indicates glycine concentration in the lower gel buffer measured relatively to the concentration of Tris
(C(Tris) = 188 mM). (B) Dependence of average bandwidth on dilution. Average bandwidth was calculated as intensity peak width at half the
peak height divided on the gel length. (C) Dependence of average Rf on glycine concentration in the gel buffer. (D) Dependence of average
bandwidth on glycine concentration. Green line indicates the bandwidth on the referent Laemmli gel. White point indicates the
unsatisfactory result because only five of six proteins bands were present. Average bandwidth was calculated as intensity peak width at half
the peak height divided on the gel length. (E) Dependence of average bandwidth on voltage applied to the gel. Point 80/150 indicated with a
star is referent—it is conventional Laemmli gel. Other gels were twice diluted and with 20% glycine added. After 200/400 point included,
extra amount of Tris-glycine buffer (TGB) was used for cooling. Gels run with cooling are indicated with blue points. Average bandwidth was
calculated as intensity peak width at half the peak height divided on the gel length.

on the new twofold diluted 20% glycine system, as shown
in Figure 5E. It is not surprising that resolution decreases
with the voltage rise and improves with cooling. Cooling
was achieved by using an extra volume of precooled TGB
running buffer. Voltages of 150 V/300 V and 300 V/600 V
with cooling were considered to have sufficient
resolution and were chosen as two options for the new
method.
4.4 Comparison with conventional
system
The method was tested on a mix of E. coli lysate proteins
and compared to the gel run according to the classical
Laemmli method. The result is presented in the Figure 6.
Although the conventional variant took 88 min to run
(Figure 6A), the “fast” (150 V/300 V) and the “very fast”

KOSHKINA et al.
F I G U R E 6 (A) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of Escherichia coli lysate. Gel conducted at
80 V/150 V, Laemmli buffer. (B) SDS–PAGE of E. coli lysate. Gel conducted at 150 V/300 V, twofold-diluted gel buffer with 20% glycine added.
(C) SDS–PAGE of E. coli lysate. Gel conducted at 300 V/600 V, extra amount of precooled Tris-glycine buffer (TGB) used for cooling,
twofold-diluted gel buffer with 20% glycine added.
(300 V/600 V + cooling) versions took 30 and 18 min,
respectively (Figure 6B,C). At the same time, it is visually
clear that the resolution is preserved. Thus, the described
method allows us to reduce the runtime of SDS–PAGE
while maintaining the resolution.
4.5 Compatibility of the technique with
Western blotting
We have assessed the compatibility of the technique with
Western blotting. A transfer to a nitrocellulose membrane
was performed and proven to be successful with Pon-
ceau staining (Figure S2). No negative impact of the gels
composition on the transfer result was detected. Antibody
staining was not performed.
5 CONCLUDING REMARKS
Current work proposes a time-, labor-, and cost-efficient
highly reproducible version of SDS–PAGE. Electrophore-
sis according to the modified method takes about 18 min,
while according to the standard method—88 min. Thus,
the electrophoretic system described herein allows us to
reduce the runtime up to fivefold, while maintaining the
resolution of the bands.
The role of glycine in the process is yet not quite clear.
It is probable that glycine molecules, uptaken by the
Cl− /Gly− boundary, increase glycine concentration above
the front and preserve buffer capacity.
Membrane transfer after the fast SDS–PAGE turned out
to be successful. Thus, the technique was proven to be suit-
able for Western blotting and could serve as a useful tool
for rapid analysis of proteomic changes. The original theo-
retical description of the electrophoretic system proposed
here is universal and can be employed to describe sys-
15222683, 0, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/elps.202300011 by National Taiwan University, Wiley Online Library on [25/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
9
tems other than Laemmli SDS–PAGE. Conclusions drawn
can be applied to similar systems, such as peptide SDS–
PAGE as an example. This should be a topic of further
investigation.
AC K N OW L E D G M E N T S
Authors thank Dr. Elena Rozhkova (Argonne National
Laboratory, IL, USA) for valuable discussion of the
manuscript.
C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors have declared no conflict of interest.
D A T A AVA I L A B I L I T Y S T A T E M E N T
The data in this study are available from the corresponding
author upon reasonable request.
ORCID
Denis L. Atroshenko https://orcid.org/0000-0003-2100-
4118
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