Protein Purification - Woe is me!!!

Curt Ashendel ashendel at aclcb.purdue.edu
Tue Oct 25 09:39:47 EST 1994


On Tue, 25 Oct 1994 13:32:30 +0000, 
Andy Hill  <afh30 at dka.sm.ic.ac.uk> wrote:
>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!

Possible positive controls:

Histone
Myelin basic protein
Protamine




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