I think you should alkylate the cysteines. I would lower the concentration
of reducing agent to maybe 20mM DTT, and after few minutes heating followed
by cooling, add final 60mM iodoacetamide. After 30min cold and dark,
purists would add further 120mM DTT to neutralize excess unreacted
iodoacetamide and so minimize alkylation of other residues (probably doesn't
matter if sole use of sample is for gel).
For most proteins, luckily, cysteines delivered onto the gel in a reducing
medium such as you describe stay reduced as the protein migrates into and
through the gel, even though the (uncharged) reducing agent remains in the
sample well. Thus monomer chains are seen in the gel. But for a few
proteins, reoxidation occurs, with appearance of multimers. In my
experience, no amount of reducing agent prevents this, but alkylation yields
a single band with lowest apparent molecular weight seen in the multimers
(or maybe even lower).
John Holt
Pasteur Merieux Connaught (soon to be Aventis Vaccines)
-----Original Message-----
From: somerville karla [SMTP:ksomer1@gl.umbc.edu]
Sent: Wednesday, May 19, 1999 2:57 PM
To: Recipients of ABRF List
Subject: hemoglobin denaturation for sds-page
Hi everyone,
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