gst fusion protein purification
rainer.strotmann at medizin.uni-leipzig.de
Thu Feb 17 09:15:49 EST 2005
> Hi, I'm affinity purifying a GST-fusion protein expressed in E.coli
> using a Glutathione-sepharose 4B matrix from Pharmacia. My problem is
> most the fusion protein remains in the column after the elution with
> glutathione. I have tested all the trouble shooting steps present in
> the pharmacia manual but anyone works. The only way to elute the
> protein is with 6 M guanidine.HCl.
> Has anyone else had a similar experience, I would be glad to hear.
> Also any suggestions on how I can elute the protein
In my hands, the elution step is extremely pH sensitive. pH 8.5 or so is
fine to elute most of the protein. Glutathione is very acidic, though,
so be sure to adjust the pH after dissolving the glutathione in your buffer.
I generally use a buffer like 50 mM Tris, pH 7.4, 100 mM NaCl. After
addition of 10 mM GSH, the pH shifts to about 3.5 and the buffer needs a
decent amount of NaOH to get back to pH 8.5.
Does this help?
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