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3 Pour the acrylamide solution into the gap between the glass
plates. Leave sufficient space for the stacking gel (the length of
the teeth of the comb plus 0.5 cm).
Using a pipette, overlay the acrylamide solution with distilled
water.
6 Pour the stacking gel mixture onto the surface of the previous
gel, place the appropriate comb into the stacking gel. Leave it to
polymerize (circa 15 minutes).
The gel can be kept at 4oC wrapped in a wet tissue and Saran
wrap.
7 Place the gel into the electrophoresis chamber and fill the
chambers with Laemmli buffer (1x).
8 Remove the combs from the stacking gel, wash the sample wells
with Laemmli buffer by pipetting the buffer up and down.
10 Heat the sample at 95oC for 5 minutes and load the samples into
the bottom of the wells.
COOMASSIE STAINING
1 Fix and stain the gel with Coomassie staining solution for 4 hours
to overnight in a slowly shaking platform. (for a faster staining,
20 minutes at 50-60oC)
WESTERN BLOT
2 Wet the filter papers and the membrane in transfer buffer (in the
case of PVDF soak before in 100% methanol).
6 Place the final two sheets of Whatman paper on the gel and cover
the sandwich with the cathode.
REFERENCES
TEMED (Tetramethylethylenediamine)