Protein Purification - Woe is me!!!

Andy Hill afh30 at dka.sm.ic.ac.uk
Tue Oct 25 08:32:30 EST 1994


Hi There!

I am trying to purify a protein which i am overexpressing in a mammalian
cell culture system. It is soluble in anionic detergents (membrane bound)
and is very basic. Hence I am trying to use a strong cation exchanger to
purify the protein away from other cellular proteins.

I am using S-Sepharose and lysing the cells in B-D-Octyl-Glucoside or
Sulfobetaine SB3-12. I have tried altering the pH and the composition of
my buffers (used Triethanolamine at ph 7.5, MES at pH 6.5). I generally
run a gradient of 0-1M salt in buffer over a 10ml column using Bio-Rad
Econo-System setup. The problem is that EVERYTHING comes out in the void
volume - i.e. nothing will stick to the column. I equilibrate the column
in the running buffer beforehand - after taking it out of the 20% ethanol
soultion.

Could anyone out there suggest anything I could try to get at least
something to stick so I know the resin works! As I am very new to this
kind of work I may have missed something simple - any comments would be
greatly appreciated!

Thanks 

Andy



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