stayve-and-irayne at worldnet.att.net
Tue Sep 28 02:00:27 EST 1999
Pierre Rodrigues <pirod at pasteur.fr> wrote in message
news:37EFA055.8AC161B6 at pasteur.fr...
> We are working on a integral membrane protein (about 10 TM segments). It
> is soluble in Triton X-100 and other detergents. But, if we heat the
> lysate at 37°C, or add high salt concentrations (0,6 M KI) it becomes
> insoluble and falls in the pellet (100.000xg) and we can see on Western
> blots high molecular weight forms which resit to SDS-PAGE...
> Does anyone knows about these solubilization artifacts (?)...? Is there
> any known effect of high salt concentrations on Triton solubilization of
> integral membrane proteins?
> Another question: if we boil SDS-PAGE samples (in laemmli buffer) and
> make Western blot on our protein, we observe it as a very high weitgh
> band which almost don't enter in the gel! Like if there is an
> aggregation. Why are some integral proteins aggregating when boiled
> before loaded on gel?
This is, unfortunately, fairly typical for some multi-spanning proteins.
Worse, there is no rule that I can figure out for which will be problemmatic
and which won't. One of the proteins we work with (betaine/GABA tranporter
BGT1) can be heated to 55'C for 3 minutes. Go much higher or for longer
times and trouble begins. Worse, if you immunoprecipitate you have to get
it off the beads before heating. Heat on the beads and no matter what the
temp it will upshift. Diluting the sample can be helpful in some instances.
Its a well known phenomenon with transporter people around here. Sometimes
urea can help, but be aware this will shift the protein up as well...just
not to the top of the gel! Good luck, it can be a real pain. Sorry I can't
be of much help.
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