acetic acid-sensitive proteins?
hkibak at leland.Stanford.EDU
Thu Jun 23 21:21:46 EST 1994
In article <CrpB8C.MAM at liverpool.ac.uk>, <agmclen at liv.ac.uk> wrote:
>Has anyone come across proteins that are very sensitive to degradation by the
>acetic acid in Coomassie stain? I am working with a protein complex that
>contains several polypeptides, most of which seem to be related by having the
>same N-terminal 10 amino acids and/or similar peptide maps. If one of the
>bands (88 kDa) is isolated pure from a Coomassie-stained SDS gel and rerun on a
>fresh SDS gel, it appears badly fragmented into ten or so smaller bands.
>Either it is very sensitive to acid staining or, perhaps, it is contaminated
>with (or is itself) an acid protease.
>I would appreciate any ideas or relevant information
One way to test this would be never to use the acetic acid in the staining
process. Instead, lower the SDS concentration in the laemli gel running
buffer to 0.04% instead of 0.1% and throw some coomassie (25 mg/L) in the
cathode (upper) buffer tank. It's a bit less sensitive than typical
coomassie staining (I would guess that the band has to be about 3 ug to
be seen this way), but much quicker and much less harsh than what you're
doing with the acetic acid. Another method is to use copper staining
as outlined by David Garfin on page 438 of Met. Enz. v. 182 (1990).
I've used both methods with sucess in cutting out bands, digesting them
with V8 and re-running them on second gels.
|| Henrik Kibak || Email: hkibak at leland.stanford.edu ||
|| Stanford University || Telephone: 408.655.6227 ||
|| Hopkins Marine Station || Fax: 408.375.0793 ||
|| Pacific Grove CA 93950 USA || Laboratory: David Epel ||
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