Hoefer SE250 - Manual (Page 25)

Appendix
The following Laemmli system is slightly modified
for use with the mini-vertical units. The Laemmli
system is the most common electrophoresis
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 electrophoresis buffer contains Tris-glycine.
All buffers contain 0.1% SDS.
Polyacrylamide gel composition is indicated by
two different percentages:
% T = total acrylamide = g (acryl + bis) × 100
100 mL
% C = crosslinker = g (bis) × 100
g (acryl + bis)
The total percent of acrylamide (% T) in the
separating gel, which can range from 5 to
20%, determines the pore size. Commonly,
the amount of crosslinker used (% C) is 2.6%.
In the following example system, 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.
•
p17
SE250 results:
Lane 1: SDS-6H, high MW
standard mixture, Sigma
™
Lane 2: SDS-7 Dalton Mark
VII-L
™ , Sigma (10 µl per lane)
Gel
12% SDS PAGE
Stained with Coomassie Blue
Running conditions
20 mA, one hour