I made a 12.5% glycine gel for protein analysis.
Materials:
12.5% separating buffer (1.5M Tris pH 8.8)
4% stacking buffer (0.5M tris pH 6.8)
Monomer solution (30% acrylamide + 2.7% bis acrylamide)
10% APS
10% SDS
Tank Buffer (250mM Tris, 192mM glycine and 0.1% (w/v) SDS)
Reducing sample buffer (2.5ml stacking buffer, 4ml 10% SDS, 2ml glycerol, 1ml b-mercaptoethanol, 35ug bromophenol blue)
Firstly, my gels took very long to polymerize (upon addition of APS and TEMED) - but my biggest problem is the running time. I have run this gel 4 times and each time it takes 5 hours and only reached halfway! I do not know what to do.
Nick Gee First 80V then 160V - however it took 8 hours only to each halfway
Run the gel at constant current (I) about 40 mAmps if one gel or 60 - 80 milliamps if two gels. Constant current results in shorter run times because at constant current your samples and the dye move at the same rate through the gel regardless of resistance of the gel which increases over the run time. So your gel will get hotter at constant current, but your samples will move at the same rate regardless of resistance of the gel. If it gets too hot just lower the current.
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