In article <firstname.lastname@example.org>,
cbookste at MEDICINE.BSD.UCHICAGO.EDU (Cres Bookstein) wrote:
> Funny you should ask. We have been discussing the unusual mobility on
> SDS-PAGE of a mammalian membrane protein with heating at 65oC vs boiling.
> This protein is not covalently bound to any lipid moiety. It has 10-12
> membrane spans and a large soluble domain. With boiling, the antigenic band
> runs at approximately 200 kDaltons. At 65oC or less, it runs at the
> molecular weight predicted by amino acid sequence. Another protein that
> behaves like this is one of the glucose transporters (Glut5?), also with
> 10-12 membrane spans and a large soluble domain and no lipid moieties.
> Our explanation is purely guess...that the boiling causes aggregation; the
> lower temperature allows unfolding without aggregation. Similar experiences
> of others will be watched with interest.
This is actually not an uncommon observation with membrane proteins. For
this reason, and for the fact that some Asp-Pro bonds appear to be labile
when boiling in SDS-PAGE sample buffer, I always have prepared my samples
for SDS-PAGE by heating at 65 degrees C for 30 minutes.
John A. Newitt, Ph.D. | <newitt at removeme.nih.gov>
National Institutes of Health | FAX: 301-402-0387
Bethesda, Maryland USA |
Please delete "removeme." from my address to reply by email.