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Binding to GST resin

ChenHA hachen at bun.com
Mon Jan 10 18:47:55 EST 2000

Cyril Privenzentzev wrote:

> Dear Netters:
> I tried to purify GST-fusion protein using Glutathione Sepharose 4B
> (Pharmacia). The protein is 30kDa DNA-glycosylase With high affinity to
> DNA. Finally, I've got a good band. However, about 80% of activity
> remains in the crude extract. Re-use of the flow-through with new batch
> of resin resulted in nothing-most of the activity goes through the
> resin.How can I improve the binding of my protein? High salt? More
> detergent?

I can't think off-hand why your protein doesn't bind to the resins, but one
thing that need to be ask: did you add something in you cell lysate (such
as benzonase) to digest away the DNA?  I'm working on a protein that has
some affinity for DNA, and not removing the DNA first seemed to have cause
a bit of a problem (the problem though is different from yours).


> Any ideas will be highly appreciated.
> Sincerely,
> Cyril VP
> --
> Dr. Cyril V. Privezentzev
> Group "Reparation de l'ADN
> CNRS UMR 8532
> Institut Gustave-Roussy
> Pavillon de Recherche II
> 39 rue Camille Desmoulins
> 94805 Villejuif Cedex
> Tel. +33 (0)1 42 11 54 04
> Fax  +33 (0)1 42 11 52 76
> ---

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