Transmembrane proteins aggregate in SDS-PAGE?

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Wed Aug 8 17:46:43 EST 2001


Dominic-Luc Webb molmed <domweb at mbox.ki.se> wrote:
>On Wed, 8 Aug 2001, Dima Klenchin wrote:
>
>> of BME, never ever heat the sample, just dissolve it at RT.
>> When I deal with NP-40/TX-100 lysates, I add 2X-3X to
>> loading buffer - these detegents strongly interfere with SDS
>> binding.
>
>Well Dima, boiling is exactly how some groups have dealt
>with the large exocytotic protein complexes and many of
>these proteins, like syntaxin and SNAP25, are membrane
>proteins, that are the object of the gel assays. Sadly,
>there is no one-size-fits-all solution.

SNARE compexes are entirtely different story unrelated to
the original question (note that I wrote "glycosylated"). SNAREs
are just a bunch of very sticky proteins. Their complexes
are nothing unusual but simply very energetically strong based
on hydrophobic interactions. Because of this, in addition to SDS, 
high temperatures are required for complete desassembly and
denaturation. 

For most proteins and their complexes however, the story is different -
they denature readily in 1% SDS at RT and their complexes fall
apart. Thus, when not dealing with special cases like SNAREs, 
boiling is necessary mostly for efficient reduction rather than 
denaturation. 

        - Dima





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