I am having a problem with completely denaturing human hemoglobin
before performing polyacrylamide gel electrophoresis. I have doubled
the usual concentration of 2-mercaptoethanol to 10% of the total sample
reducing buffer (which contains 12.5% 0.5M Tris-HCl pH 6.8, 10% glycerol,
2% SDS, and .02% bromophenol blue). I have been boiling the samples for
10 minutes and still when I run the gel I get a dimer peak at 32,000 MW.
I have even tried adding EDTA and urea (separately, then together) to help
denature/unfold the protein.
Are there any suggestions as to what else I may be able to do to solve
this problem?
Karla Somerville-Armstrong
UMBC
1000 Hilltop Circle
Baltimore, MD 21250
ksomer1@gl.umbc.edu