elution of GST fusions

[Marc Champagne]

In article <33AC9DAD.2BCF at BIOSCI.MBP.MISSOURI.EDU>,
NOTHWEHR at BIOSCI.MBP.MISSOURI.EDU wrote:

> We have successfully expressed a GST fusion protein in E. coli.  The
> protein of interest is not fused to the C-terminus of GST as is usually
> the case but rather to the N-terminus. Our problem is that we have not
> been able to elute the fusion protein from glutathione agarose using
> reduced glutathione even after prolonged incubation periods at room
> temp.  In contrast GST alone elutes just fine under the conditions we
> have used.  Of course our fusion protein does elute under denaturing
> conditions such as 100 deg. in the presence SDS-PAGE laoding buffer. 
> Does anyone know of any other conditions involving reduced glutathione
> or a related molecule that would specifically dissociate GST fusions
> from glutathione agarose?  Thanks.

Have you tried 50 mM Glutathione, and have you verified the pH?  pH is critical.

m

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