In the past, I have always reduced my sample prior to running on SDS PAGE
and followed the standard protocols for reduction/alkylation of gel slices
to obtain alkylated cysteines if required. I now have the need to alkylate
just prior to SDS PAGE.
Would those out there who routinely reduce and alkylate their samples PRIOR
to SDS PAGE care to share their protocols? I have tried to glean a few
protocols from the literature and have tried the following:
Sample in standard SDS sample buffer (60mM Tris pH 6.8, 2% SDS)
made 10mM DTT by addition of 1M DTT (in water)
100 degrees celcius for 10 minutes
cooled to room temperature
made 50mM iodoacetamide by addition of 1M iodoacetamide (in water)
room temperature in dark for 15 minutes
made 2% mercaptoethanol to quench iodoacetamide by addition of neat
mercaptoethanol.
I notice the bromphenol blue in the sample buffer showing a mild drop in pH
during the alkylation step and it goes quite yellow some time after the
addition of the mercaptoethanol and needs quite a few microlitres of
concentrated Tris to get the pH back to normal running levels.
Is this pH change normal?
Regards....Ken
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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
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