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eluting gst fusion proteins


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#1 kmb

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Posted 16 December 2010 - 01:06 PM

Hi. I'm trying to elute GST-tSNARE (made up of GST-syntaxin1a and SNAP-25) from glutathione sepharose beads. We tried adding 20mM glutathione in our regular Hepes buffer (20mM HEPES-KOH pH 8.0, 150mM KCl, 5mM DTT, 0.05% n-octylglucoside) that we use to purify our proteins at different elution time points.

What we found was that
1) most of our protein wasn't being eluted (based on a protein assay most apparently stayed on the beads)
2) elution seemed to disrupt binding between syntaxin and SNAP-25 such that when we ran the get, we only saw a prominent band for SNAP-25 whereas the GST-syntaxin wasn't present regardless of elution time.

Does anyone have any thoughts as to how to deal with these two problems? I've considered increasing the concentration of glutathione to see if that would get the protein off the bead but I am not sure why the complex is being disrupted. Most of the protocols to do gst elutions seem to use Tris instead of Hepes. Any thoughts as to if this might make a difference in this case?

Thanks!

#2 mdfenko

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Posted 17 December 2010 - 11:49 AM

do you see any of your protein in the elution fraction? if so, then you can stop the flow of the column after elution starts (or you are sure that the elution buffer has completely displaced the wash buffer). wait an hour or longer then restart the flow. this will enhance elution.
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