Hoefer SE400 - Manual (Page 37)

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Appendix A. Laemmli System Gels
Table 4. Laemmli gels — final concentrations
Electrophoresis
Resolving gel Stacking gel buffer
Acrylamide conc. 10% T*, 2.6% C 4% T, 2.6% C
Tris-Cl 0.375 M 0.125 M
Tris-Glycine 0.025 M Tris base
0.192 M glycine
pH 8.8 6.8 ~8.3
SDS 0.1% 0.1% 0.1%
APS † 0.05% w/v 0.05 – 0.1% w/v
TEMED ‡ 0.05% v/v 0.05 – 0.1% v/v
* To achieve any other desired final concentration, adjust the acrylamide
stock and water volumes. Volumes for different concentrations are
listed in Table 5.
† Ammonium persulfate.
‡ Tetramethylethylenediamine
The Laemmli system is the most common electropho-
resis protocol for SDS-denatured proteins. The leading
ion in this discontinuous buffer system is chloride and
the trailing ion is glycine. Accordingly, the resolving
gel and the stacking gel contain Tris-Cl buffers (of
different concentration and pH), and the electropho-
resis buffer contains Tris-glycine. All buffers contain
0.1% SDS.
Polyacrylamide gel composition is indicated by two
different percentages:
The total percent of acrylamide (%T) in the resolving
gel, which can range from 4 to 20%, determines the
pore size. Commonly, the amount of crosslinker used
(%C) is 2.6%. In the following system example, the
resolving gel composition is 10%T, 2.6%C, which
results in a medium pore size. The stacking gel
composition is 4%T, 2.6%C. The %T in the stacking
gel is lower because a larger pore size is required.
Caution! Acrylamide is a
neurotoxin. Always wear gloves
while handling in any form and
wear a mask while weighing the
powder. Never mouth pipette
the solution.
g(bisacrylamide)
g(acrylamide + bisacrylamide)
%C = × 100
g(acrylamide + bisacrylamide)
100 ml
%T = × 100