Transmembrane proteins aggregate in SDS-PAGE?

Dominic-Luc Webb molmed domweb at mbox.ki.se
Thu Aug 9 08:42:38 EST 2001


On 9 Aug 2001, renaud leonard wrote:

> perhaps should you lyse your cells in presence of SDS and beta
> mercaptoéthanol and at 37)C to limit agrégation.
>

Sadly this often fails for tight complexes. "Aggregates"
are not likely to involve much more than weak interactions
that should be eliminated by the above protocol. But a
lot of complexes of functionally associated proteins
often involves intertwined helixes with very large amount
of hydrogen bonds and interlocking due to coils
wrapping around themselves. I know that the exocytotic
proteins are notorious for making SDS-mercaptoethanol
resistant complexes that I have not been very successful
in removing even by boiling and the reason is clearly
suggested in the published 3D structures of these complexes.

Once these complexes are split, they do not reform in
SDS. On the other hand, you need a lot of heat to melt
them and I suspect the SDS/DTT buffers pull down the
boiling point too much to reach a temperature to melt
these complexes. I suggested to the original poster to
try boiling in a weak inverted (intracellular-like)
buffer and then add SDS/DTT, in an effort to actually
reach 95-98 degrees (trying not to get the tubes to
burst open).

Microwave has been used by several groups in studies of
exocytotic proteins. I'll bet if you look at some of
the excellent work done by Richard Scheller or Reinhard
Jahn examining the exocytotic proteins you will find
a nice illustration of just how tough these complexes
are and just how much is needed to break them. In fact,
I think it was Reinhard Jahn that presented a very
telling melting curve for SNAP25 and syntaxin a couple
years ago. OTOH, there was a very steep curve and you
really needed to get close to 100 C to break these
complexes decently. You have to check their papers as
this is not my specialty (I just know about their work
and read their papers once in a while).



Cheers,



Dominic




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