Binding to GST resin

Scott Robson scott at nmr1.UCHC.EDU
Mon Jan 10 16:41:23 EST 2000


The only thing I can think of is a two step problem...

a) Your crude extract has so much GST fusion protein in it that the column
is saturated and can;t bind the 80% of activity that flows though. However
this should rebind to a clean column as you have tried... unless.....

b) you have contaminated your flow through with glutathione from a washing
procedure.

If you are sure you have no free glutathione in your flow through then I
can only think of one other problem (purely speculative)...

20% of your protein is not binding DNA. The other 80% for reasons best
left to strutural biologists somehow effects GST binding to GSH preventing
it from binding. While it remains bound to DNA it might dimerise in such a
way that two GST domains interfer with each other preventing GSh binding.

Salt may help (give it a go, NaCl is cheap) you can also try diluting your
cell lysate and applying the protein at a lower concentration (takes
longer I know, but time is relatively cheap too). Depending on how you
prepare your lysate you might like to try and break down the DNA even
further. Add some DNase or something. Try a lysis buffer with and without
5mM EDTA.

Goodluck!

Scott

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?
> 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
> FRANCE
>
> Tel. +33 (0)1 42 11 54 04
> Fax  +33 (0)1 42 11 52 76
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