Thrombin Cleavage of GST

[Guan Zhu]

Evan,

If you have to immunize (or have already immunized) rabbits with
GST-fusion protein, you may express only the GST (or even any other
GST-fusion protein if handy, but not the fusion protein of your interest)
and make a GST-agarose column, let the antiserum go through the column to
absorb the anti-GST antibody from the antiserum. Run a immuno-blot
analysis to assure the result.

We have expressed a small peptide fused with GST which was difficult to be
eluted after thrombin cutting. The  problem was then solved using above
approaches.

Good luck!


In article <32237D17.835 at wheel.dcn.davis.ca.us>, Lyle Najita
<ijiwaru at wheel.dcn.davis.ca.us> wrote:
> > Hi,
> >    I have a fusion protein tagged to GST and am trying to cleave the GST
> > fusion partner off with thrombin. It does not seem that cleavage is
> > efficient if at all. Could anyone recommend a protocol that they know
> > work? I would be extremely grateful.
> > 
> > Another thing, is it necessary to cleave the GST off if wanting to inject
> > into rabbits for antisera to the recombinant. I subsequently want to use
> > the fusion protein (uncleaved) for ELISA. Will I get too high a
> > background in the ELISA to the GST? Does anyone recommend raising
> > antisera specifically against the recombinant?
> > 
> > Thanks in advance,
> > Evan Burkala
> > <eburkala at central.murdoch.edu.au>

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Guan Zhu, PhD
Wadsworth Center, NYS DOH       Phone (518) 474-2187
David Axelrod Inst, Rm 3063     Fax   (518) 473-6150
P. O. Box 22002
Albany, NY 12201-2002           guan.zhu at wadsworth.org
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