The basic SDS-PAGE method is based on that described by Laemmli (10,11) and as an aid the 12.5% polyacylamide gel, is described in Subheading 2.4. (see also Note 12). SDS polyacrylamide gels are electrophoresed in running buffer (Note 13). Gels containing SDS separate on the basis of molecular weight, and it is possible to calibrate the gel using a sample of appropriate marker proteins; these are commercially available as prestained, or 14C-labeled markers, which allow them to be visualized by autoradiography or on immunoblots.
1. The resolving gel is poured (adding the TEMED and ammonium persulfate last), and the surface is flattened by overlaying with a small amount of butan-1-ol.
2. After polymerization the butan-1-ol is drained off, and the stacking gel poured on top of the resolving gel.
3. To the sample, an equal volume of 2X dissociation buffer is added and boiled for 3 min prior to loading onto the gel (see Fig. 1).
After electrophoresis, the gels can be fixed and stained with Coomassie blue to visualize the proteins if the gel is not to be immunoblotted (see Note 14). The gels are fixed for 30 min, and then stained with Coomassie blue for 4 h, followed by destaining in "fixing solution," which can be accelerated at 50°C.
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