Gel filtration vs gradient
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri Nov 26 14:04:05 EST 1999
:We are trying to look at oligomers of an integral protein. We compared
:gel filtration (by HPLC) to sucrose gradients and obtained opposite
:results: in HPLC, most of the protein was eluted on a 300-400 kDa range
:whereas gradient elution was around monomer size!
:As our protein is an integral membrane protein and has 2/3 of its lenght
:hydrophobic, could there be lipids still interacting with it and make it
:float on gradients?
:We did this experiments on Triton x-100 and CHAPS and did a 100000xg
:If anyone has some explanations (even partial!) it would be great...
Gel-filtration measures geometry of the molecule, while gradients measure
density and/or mass (depending on kind of gradient you do). As such, there
could be little correlation between the two. Presence of detergents
in both types of experiments only complicate things. I don't think
unambigous conclusion can be reached in either case.
I'd suggest using crosslinkers (better several of them at a broad range
of concentrations each) and running SDS PAGE. (A control with a protein
that does not oligomerize is essential). This, IMHO, will provide you with
better understanding what's going on.
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