electrophoresis
tech note 5910
Mini-PROTEAN TGX ™ Precast Gel: A Gel for SDS-PAGE with
Improved Stability — Comparison with Standard Laemmli Gels
®
Tom Berkelman, Shane Petersen, Chengjun Sun, and Sean Cater,
Bio-Rad Laboratories, 6000 James Watson Drive, Hercules, CA 94547
Introduction
SDS-PAGE is a widely used and versatile method that
separates proteins according to size and allows estimation of
molecular weight. SDS-PAGE involves treatment of the sample
with SDS and a thiol reductant (usually b-mercaptoethanol
or dithiothreitol). SDS is an anionic detergent which forms
complexes with proteins giving them a rod-like shape and a
uniform charge to mass ratio. The reductant cleaves disulfide
bonds between and within proteins allowing complete
denaturation and dissociation. Heat treatment in the presence
of SDS and reductant effectively eliminates the effects of
protein conformation and native charge on electrophoretic
mobility so the migration rate depends primarily on molecular
weight. Molecular weight is estimated by comparison to
standards of known molecular weight.
The most commonly used SDS-PAGE method is the
so-called Laemmli system, which was first published in
1970 (Laemmli, 1970). This system relies on a discontinuous
buffer system. Two ions of differing electrophoretic mobility
(glycinate and chloride) form a moving boundary when
voltage is applied. Proteins have an intermediate mobility,
causing them to concentrate or “stack” into a narrow zone
at the beginning of electrophoresis. The stacking effect is
responsible for the high resolving power of the Laemmli
system. The sample is loaded in a relatively broad zone, and
the moving boundary concentrates the proteins into sharp
bands prior to separation. As the boundary moves through
the gel, the sieving effect of the polyacrylamide gel matrix
causes different proteins to move at different rates.
The percentage of polyacrylamide controls the separation
characteristics of the gel. Higher percentage gels have a more
pronounced sieving effect and are best suited for separations
of relatively low molecular weight proteins. Lower percentage
gels are better suited for separations of larger proteins. Gels
can also be cast with a polyacrylamide percentage gradient
to give a broader separation range.
Commercially prepared Laemmli system gels typically
have a shelf life of only a few months and separation
performance degrades steadily over time. Despite the short
shelf life of most precast gels, Laemmli system SDS-PAGE
is very widely used. It is regarded as the “gold standard”
of SDS-PAGE techniques due to its resolution, robustness,
ability to accurately estimate molecular weight and
compatibility with a wide variety of sample types and sample
solution compositions. Laemmli system SDS-PAGE also
has the advantage of only requiring relatively common
and inexpensive buffer components (Tris and glycine).
Bio-Rad’s Mini-PROTEAN TGX gels for SDS-PAGE are
based on a modification of the Laemmli system that gives
significantly improved stability and performance over time.
Mini-PROTEAN TGX gels retain separation characteristics
similar to Laemmli gels and use standard running and
sample buffers. In this study, the performance of TGX gels
and standard Laemmli system precast gels are compared
following storage at 37ºC to mimic accelerated assay
conditions, and the separation performance and accuracy
of molecular weight estimation of both gel systems
are presented.
Table 2. Proteins used for molecular weight estimations.
Protein
Size*, kD
Rabbit myosin
200
Human a-macroglobulin161
E. coli b-galactosidase116
Rabbit phosphorylase b
97
Human apotranferrin
77
Chicken conalbumin
76
Bovine serum albumin
66
Bovine glutamic dehydrogenase
55
Chicken ovalbumin
45
Protein
Size*, kD
Equine alcohol dehydrogenase
40
Rabbit aldolase
39
Enterobacter b-lactamase39
Rabbit glyceraldehyde phosphate dehydrogenase
36
Bovine carbonic anhydrase
31
Bovine trypsinogen
24
Bovine b-casein23.6
Bovine myelin basic protein
18.3
Bovine a-lactalbumin14.2
* The indicated molecular weights were either derived from gene sequence data or provided by the supplier.
To evaluate accuracy of molecular weight estimation of
the two gel systems, eighteen different purified proteins
(Table 2) were run on both TGX and Laemmli gels of two
percentages (10% and 4–20%). A lane of Precision Plus
Protein unstained standards (Bio-Rad) was also run on each
gel. Standard curves were generated by plotting the logarithm
of the molecular weight of each standard vs. its R f and the
molecular weight of each purified protein was estimated
using a linear fit to the standard curve for each gel. With both
gel percentages, molecular weight estimated by SDS-PAGE
was more accurate using Mini-PROTEAN TGX gels than
using Laemmli gels, as judged by the correlation coefficient
between the molecular weight estimated by SDS-PAGE and
the true molecular weight (Figure 6).
A. 10% TGX
Estimated mass, kD
250
200
Correlation coefficient = 0.994
Regression coefficient = 1.010
B. 10% Laemmli (Ready Gel)
250
200
150
150
100
100
50
50
0
0
0 50 100150200250
True Mass, kD
C. 4–20% TGX
Estimated mass, kD
250
200
Correlation coefficient = 0.972
Regression coefficient = 0.867
Correlation coefficient = 0.994
Regression coefficient = 1.045
0
D. 4–20% Laemmli (Ready Gel)
250
200
150
150
100
100
50
50
0
50 100 150 200250
True Mass, kD
Correlation coefficient = 0.974
Regression coefficient = 0.708
The key feature of the TGX gel is better stability than
standard Laemmli system gels. The effect of incubation at
37ºC well illustrates the superior stability and shelf life of the
Mini-PROTEAN TGX gel. A longer shelf life confers benefits
beyond not needing to order gels as frequently. Reproducibility
of separation is also enhanced. The separation characteristics
of Laemmli gels change continually, and this change is not
observed in Mini-PROTEAN TGX gels that are used before
their expiration date.
In addition to improved stability, Mini-PROTEAN TGX gels
have demonstrable advantages in terms of the both quality
of separation and the ability to estimate molecular weight
accurately. More consistent band width and shape allow
better quantitation by densitometry. Since one of the primary
purposes of SDS-PAGE is to estimate the molecular weight
of proteins, any improvement in the linearity of separation
behavior and the accuracy of molecular weight estimation is
also a significant advantage.
References
Laemmli UK (1970). Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227, 680-185.
Coomassie is a trademark of BASF Aktiengesellschaft.
Information in this tech note was current as of the date of writing (2009) and
not necessarily the date this version (rev A, 2009) was published.
0
0 50 100150200250
True Mass, kD
Conclusions
Mini-PROTEAN TGX gels are based on a modification of
the Laemmli system for SDS-PAGE. The gel chemistry
was designed to have similar separation characteristics to
the Laemmli gel and to use the same running and sample
buffers. Similar separation patterns should simplify adoption
of the Mini-PROTEAN TGX system by users familiar with the
Laemmli system.
0
50 100 150 200250
True Mass, kD
NOTICE TO PURCHASER: LIMITED LICENSE
*T his product is sold under license from Life Technologies Corporation,
Carlsbad, CA for use only by the buyer of the product. The buyer is not
authorized to sell or resell this product or its components.
Fig. 6. Accuracy of molecular weight estimation between Mini-PROTEAN
TGX and Laemmli gels. Eighteen different purified proteins (Table 2) were run
on Mini-PROTEAN TGX gels and Ready Gel Tris-HCl gels (Laemmli gels) along
with Precision Plus Protein unstained standards. Both 10% and 4–20% gels
were evaluated. The molecular weight of each purified protein was estimated
from plots of Log MW vs. molecular weight of the standards after SDS-PAGE.
This estimated molecular weight was plotted against the actual mass.
© 2009 Bio-Rad Laboratories, Inc.
Bulletin 5910
Methods
Samples
Samples used included broad range unstained SDS-PAGE
standards (Bio-Rad Laboratories, Inc.), Precision Plus
Protein™* unstained standards, E. coli lysate (Bio-Rad), mouse
serum, soybean extract (prepared by hydrating soybeans
overnight in aerated water and grinding in nine volumes of
50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM PMSF, 40 µM
bestatin, 10 µM leupeptin, 10 µM E64 and removing insoluble
material by centrifugation).
Purified proteins used in this study were purchased from
Sigma Aldrich Co.
Electrophoresis and Imaging
Electrophoresis was performed on 7.5%, 10%, 12%, 4–15%,
and 4–20% Mini-PROTEAN TGX precast gels and on
Ready Gel® Tris-HCl precast gels of the same percentages.
Samples were prepared in Laemmli sample buffer containing
5% b-mercaptoethanol. Precision Plus Protein unstained
standards were loaded as provided. Gels were run using
the Mini-PROTEAN Tetra electrophoresis cell at 200 V
until the dye front reached the bottom of the gel. Both types
7.5%
TGX of gels were run using Tris/Glycine/SDS running buffer
(25 mM Tris, 192 mM glycine, 0.1% [w/v] SDS). Gels were
stained with Bio-Safe™ Coomassie stain (Bio-Rad) and
imaged on a Molecular Imager ® GS-800™ calibrated
densitometer (Bio-Rad).
Results
Comparison of Migration Patterns Between Mini-PROTEAN TGX
and Laemmli Gels
The choice of gel percentage is determined by the molecular
weight range of interest. Mini-PROTEAN TGX gels are
designed to have separation characteristics similar to the
corresponding percentage of a standard Laemmli gel
(Ready Gel precast gels) and therefore offer a predictable
alternative. Similarity of separation behavior was evaluated
by running natural protein standards on Mini-PROTEAN TGX
gels and Laemmli gels of the same percentage. Migration
patterns were indeed found to be very similar between the
two different gel types (Figure 1). Whereas the mobilities of
individual standards differ slightly between the two gel types,
separation ranges were very similar between Mini-PROTEAN
TGX gels and Laemmli gels of the same percentage (Figure 1).
10%
TGX Ready Gel
116
97
116
97
116
97
66
66
66
45
45
45
31
31
31
21.5
14.4
21.5
4–15%
TGX
4–20%
TGX
Ready Gel
200
116
97
66
66
45
45
21.5
14.4
6.5
Ready Gel
200
116
97
31
Ready Gel
200
200
200
12%
TGX
Ready Gel
31
21.5
14.4
6.5
Fig. 1. Comparison of migration patterns of natural protein standards between Mini-PROTEAN TGX and Laemmli gels. Broad range SDS-PAGE standards
were run in triplicate on Mini-PROTEAN TGX gels and Ready Gel Tris-HCl gels (Laemmli gels) of the percentages indicated. The standards used are: rabbit myosin
(200 kD), E. coli b-galactosidase (116 kD), rabbit phosphorylase b (97 kD), bovine serum albumin (66 kD), chicken ovalbumin (45 kD), bovine carbonic anhydrase
(31 kD), soybean trypsin inhibitor (21.5 kD), chicken lysozyme (14.4 kD), bovine aprotinin (6.5 kD). Gel migrations were aligned at the 200 kD to demonstrate the
migration pattern.
© 2009 Bio-Rad Laboratories, Inc.
Bulletin 5910
10% Mini-PROTEAN TGX gels stored at 37ºC
12 34 5 6 78 9
10
1234 5 678910
12345 6789
10
0 day
6 days
12 days
1 2 34 5 6 78 910
1 2 34 5 6 78 910
1 2 34 5 6 78 910
0 day
6 days
12 days
250
150
100
75
50
37
25
20
10% Laemmli gels stored at 37ºC
250
150
100
75
50
37
25
20
Fig. 2. Comparison of separations on Mini-PROTEAN TGX and Laemmli gels following storage at 37ºC. Freshly prepared 10% Mini-PROTEAN TGX gels
and Ready Gel Tris-HCl gels (Laemmli gels) were incubated at 37ºC for the amount of time indicated. Following the treatment, they were loaded as follows:
Lanes 1 and 9, Precision Plus Protein unstained standards; lanes 2 and 10, broad range SDS-PAGE standards; lanes 3-5, E. coli lysate; lanes 6-8, mouse serum.
Comparison of Stability Between Mini-PROTEAN TGX
and Laemmli Gels
Separation performance in standard Laemmli gels degrades
over time due to decomposition of the polyacrylamide matrix.
Mini-PROTEAN TGX gels have been designed to address
this shortcoming and have a significantly longer shelf life.
The effect of prolonged storage was accelerated by
incubation at 37ºC and stability was assessed by running
standards and samples following 0, 6, and 12 days of
incubation. There was no discernible effect of 37ºC
treatment on the Mini-PROTEAN TGX gels, but storage
at elevated temperature had a marked negative effect on
the performance of the Laemmli system gels (Figure 2).
A Mini-PROTEAN TGX gel stored under recommended
conditions (4ºC) for 12 months still exhibits high resolution
separation (Figure 3).
© 2009 Bio-Rad Laboratories, Inc.
1234
Fig. 3. Separation on Mini-PROTEAN TGX gel stored for 12 months
at 4ºC. A 4–20% Mini-PROTEAN TGX gel was kept at 4ºC prior to running
standards and samples. Lane 1, Broad range SDS-PAGE standards;
lanes 2–4, E. coli lysate.
Bulletin 5910
Comparison of Band Sharpness Between Mini-PROTEAN TGX
and Laemmli Gels
Linearity and Accuracy of Molecular Weight Estimation
on Mini-PROTEAN TGX and Laemmli Gels
Accuracy of molecular weight estimation and quantitative
densitometry are best when samples run as straight
consistent lanes and form regular symmetrical bands.
When an identical sample was run on both Mini-PROTEAN
TGX and Laemmli system gels, the protein bands on the
Mini-PROTEAN TGX gel were consistently about the same
width as the loading well, whereas band spreading was
observed with the Laemmli gel. In general, bands were
straighter and more regular with the Mini-PROTEAN TGX gel
than with the Laemmli gel (Figure 4).
Molecular weight is estimated using a standard curve that is
generated by plotting the logarithm of the molecular weight of
each standard vs. its R f (R f is the ratio of the distance traveled
by the protein to the distance traveled by the electrophoretic
front). The standard curve approximates a straight line and
molecular weight is most conveniently estimated using a
linear fit to the standard curve. The linearity of the standard
curve will therefore influence how accurately molecular weight
may be estimated. Mini-PROTEAN TGX gels and Laemmli
gels were evaluated with respect to linearity by running
standards made of natural proteins and comparing curves
of Log MW vs. R f between same percentage gels of each
gel type. Regression lines were drawn and R2 values were
calculated (Figure 5 and Table 1). In all cases except for the
4–15% gradient gel, the Mini-PROTEAN TGX gels exhibited
better linearity than the Laemmli gels. The difference in
linearity was more significant among the single percentage
gels (7.5%, 10%, and 12%) than among the gradient gels.
Mini-PROTEAN TGX 10%
Ready Gel 10%
Table 1. Linearity between Mini-PROTEAN TGX and Laemmli gels
using natural protein standards.
R 2*
10%
1.2
2.4
Log MW
2.22.2 2.2
4 0.2 0.6 0.4 0.8 0.6 1.0 0.8
2.02.0 2.0
Rf
R
f
1.8
1.8
1.8
Log MW
Log MW
2R
R1.2
=2 0.9894
= 0.9894
R2 = 0.9894
2 =2 0.9621
R1.0
R = 0.9621
R2 = 0.9621
1.0
1.0
0
0.2
0
Rf
1.2
2.2
1.0
1.8
1.8
R
f
1.8
2.2
R
1.8
1.8
f
1.8
1.61.6
1.6
1.61.6
1.6
1.41.4
1.4
1.41.4
1.4
1.21.2
1.2
1.21.2
1.2
1.21.2
1.2
1.01.0
1.0
1.01.0
1.0
1.01.0
1.0
0.2
0.40.4
0.40.60.6
0.6
0.80.8
0.81.01.0
1.0
00
4-15%
RR
R
f
f
00.20.2
f
2.0
4–20%
R2 = 0.9928
R2 = 0.9957
R2 = 0.9928
R2 = 0.9957
1.5
2.52.5
2.0
2.0
1.5
Log MW
Rf
Rf
1.51.5
1.0
2.5
2.02.0 2.0
0 0.4 0.2 0.6 0.4 0.8 0.6 1.0 0.8
Log MW
Log MW
0.2
2.5
4-15%
4-15%
4-15%
1.0
0.5
2.5
Log MW
2.0
0.40.60.6
4-20%
Rf Rf
Log MW
2.5
0.2
0.40.4
00.20.2
0.2
0.40.4
0.40.60.6
Rf Rf
0.6
0.80.8
0.8
1.01.0
Rf
R2 = 0.9974
R2 = 0.9902
4-20%
4-20%
4-20%
2.52.5
0.2
2.5
2.02.0 2.0
0 0.4 0.2 0.6 0.4 0.8 0.6 1.0
Rf
1.5
00
1.0
1.0
1.5
1.0
0
0.8
1.01.0
R2 = 0.9974
R2 = 0.9902
R2R=2 0.9928
= 0.9928
R2 = 0.9928
R2R=2 0.9957
R2 = 0.9957
= 0.9957
0.5
0.5
1.0
0.6
0.80.8
4-20%
Rf
Log MW
00.20.2
R2R=2 0.9815
= 0.9815
R2 = 0.9815
R2R=2 0.9277
R2 = 0.9277
= 0.9277
0.4 0.2 0.6 0.4 2.0
0.8
2.0 0.6
2.01.0 0.8
1.4
Log MW
Log MW
0
2.4
2.22.2
1.6
2.5
0
0.2
Rf
4–15%
0.5
1.0 0
2.42.4
1.41.4
4-15%
1.0
1.2
R2R=2 0.9847
= 0.9847
R2 = 0.9847
R2R=2 0.9527
R2 = 0.9527
= 0.9527
1.0
2.4
12%
1.61.6
00
1.5
1.4
2.22.2
0.4 0.2 0.6 0.4 2.0
0.8
2.0 0.62.01.0 0.8
12%
12%12%
1.4 10%
2.42.4
Log MW
Log MW
2.42.4
1.4
1.8
* R2 values
from the data shown in Figure 5 are compared.
1.610%
1.6
10%10%
Log MW
1.4
1.6
Ready Gel precast gels
7.5% 0.98940.9621
10%
0.98470.9527
2.4
R2 = 0.9815 R2 = 0.9815
12%
0.98150.9277
R2 = 0.9277 R2 = 0.9277
2.2
4–15% 0.99280.9957
2.0
4–20%0.99740.9902
Log MW
Log MW
7.5%
7.5%7.5%
7.5%
Log MW
1.6
Log MW
Log MW
Log MW
Fig. 4. Comparison of soybean extract separation between
2.4 system gels. Soybean
Mini-PROTEAN TGX2.4
and Laemmli
extract (~10 µg)
2.4
2
R = 0.9894 R2 = 0.9894
R2 = 0.9847 R2 = 0.9847
run on
10%
Mini-PROTEAN
TGX
0.9621
0.9621
R2 =was
R2 =
R2Tris-HCl
R2 = 0.9527 2.2
= 0.9527 gel.
2.2
2.2 and 10% Ready Gel
A single lane from each gel was compared. A protein doublet of ~90 kD was
2.0
2.0
selected from each gel and its image was enlarged to illustrate the difference 2.0
1.8
1.8
1.8
in band appearance.
Mini-PROTEAN
12%
12% TGX gels
Log MW
10%
7.5%
Log MW
Log MW
7.5%
Gel Percentage
Rf
1.51.5
R2R=2 0.9974
= 0.9974
R2 = 0.9974
R2R=2 0.9902
R2 = 0.9902
= 0.9902
0.8
1.0
1.5
Fig. 5. Linearity between Mini-PROTEAN TGX and Laemmli system gels. Broad Range SDS-PAGE standards were run on Mini-PROTEAN TGX gels and
Ready Gel Tris-HCl gels (Laemmli
of the percentages indicated. The standards are: rabbit
1.01.0 system)
1.0
1.0myosin
1.0 1.0(200 kD), E. coli b-galactosidase (116 kD), rabbit
phosphorylase b (97 kD), bovine serum albumin (66 kD), hen ovalbumin (45 kD), bovine carbonic anhydrase (31 kD), soybean trypsin inhibitor (21.5 kD), hen lysozyme
(14.4 kD), bovine aprotinin (6.5 kD). Log MW vs. Rf is compared between gel types of the same percentage on the same set of axes. Each value is an average of
0.50.5 0.5
0.50.5 0.5
nine Rf values (from three lanes run on three gels). The Rf of bovine aprotinin was not used in this study because this small protein is consistently observed to run
00
00.20.2 0.2
0.40.4 0.4
0.60.6 0.6
0.80.8 0.8
1.01.0 1.0
00
00.20.2 0.2
0.40.4 0.4
0.60.6 0.6
0.80.8 0.8
1.01.0 1.0
anomalously. ◆, Mini-PROTEAN TGX gels; n, Ready Gel Tris-HCI gels.
Rf Rf
© 2009 Bio-Rad Laboratories, Inc.
Rf
Rf Rf
Rf
Bulletin 5910
1.0
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 31 3689 6600
Canada 905 364 3435 China 86 20 8732 2339 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 69 65
Germany 089 31 884 0 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050
Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500
Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723
Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000
Life Science
Group
Bulletin 5910 Rev B
US/EG
10-1203
0810
Sig 1109