Bio-Rad Sequi-Blot™ PVDF Membrane User Manual
Page 7
5.
Allow the gel, including stacker, to poly-
merize completely. Let the cast gel stand
for 24-72 hours at room temperature prior
to use.
6.
Add 11.4 mg/l (0.1 mM) thioglycolate to
the upper running buffer prior to elec-
trophoresis to scavenge reactive com-
pounds left in the gel which cause
N-terminal blocking.
7.
Include 5 µg of a sequence standard (i.e.
myoglobin or ß-lactoglobulin A) as a con-
trol in one lane.
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proteins with a blocked amino terminus, are
reviewed in bulletin 2212.
Follow your standard procedure for
Laemmli SDS-PAGE, observing the changes
outlined below (solution recipes are included
after the blotting section):
1.
Use reagents and solvents of the highest
purity. Use of Bio-Rad’s electrophoresis
reagents without further purification is rec-
ommended.
2.
Filter gel solutions, except running buffer,
with a 0.45 micron filter and store at 4 °C.
Store SDS stock solution at RT.
3.
Solubilize samples with 2x or 5x solubiliz-
ing buffer with sucrose. Do not use urea in
the solubilizing buffer.
4.
Heat samples with solubilizing buffer at
37 °C for 10-15 minutes prior to loading
onto the gel. Do not heat samples at 100 °C.
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