Tracy,
Is your protein too large to fold correctly if you simply let the Sulfhydryls oxidize after
removing the DTT by dialysis? I would try that. I would also try the analog in case that works
better. (I assume you have one all picked out - I don't know of any off-hand.) Alternatively, can
you change your construct to a His tag instead of the GST fusion and avoid the sulfhydryl problem
that way? Good luck. Laura
tmitch1 wrote:
> Hello. I was wondering if anyone has used a glutathione analog in which the sulfhydral group
> is blocked to elude bound GST-fusion proteins from a GSH-Sepharose Chromatography Column
> (Pharmacia). Reduced glutathione (GSH) is currently used, but it binds to our peptides
> via two disulfide bonds to two cysteines in our target peptide. DTT removes the GSH, but it
> produces the target peptide with reduced cysteine residues. We suspect that the cysteines
> form a native disulfide bond in the absence of GSH and DTT. Any suggestions? Thanks for your
> time.
>> Tracy Mitchell
> Loyola University of Chicago
>tmitch1 at orion.it.luc.edu
