John,
I agree that pre-gel alkylation would represent a good approach to this
problem. We routinely use pre-gel acrylamide alkylation on our cysteines
(alkylating with 1% acrylamide in sample buffer after boiling in 10mM DTT).
One comment I would make is that most people forget that SDS sample buffer
is usually pH 6.8 whereas the reaction kinetics of reduction and alkylation
are much more favoured at pH 8.5. We have been doing these alkylations in
regular sample buffer adjusted to pH 8.5(thanks to Steve Tindall for
suggesting this) and find the alkylations are quite complete at 30 minutes
and the gels run just fine.
Regards...Ken
>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
>
>
>
********************************
Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org
***********************************