GST pull down

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Mon Mar 22 12:55:04 EST 1999


:I need suggestions and or references concerning using a GST tagged protein
:(immobilized on a Pharmacia glutathione-sepharose column) to pull out protein
:binding partners from a plant cell lysate.
:
:Any ideas will be greatly appreciated, thanks

This in very common, flawed and overused method. Most of the time, 
people run E.coli lysate from bugs overexpressing the protein over
Glutation-Sepharose, wash well and incubate resulting "beads" 
with extracts of interest. The conditions typically used are as
in your typical solid phase immunoprecipitation exp. After that, 
you boil the stuff in SDS and analyse protein by electrophoiresis.

I would caution against puttinjg too much weight onto this unless
you have significant amounts of interacting partner, fairly high
affinity interaction, and an array of good negative and positive controls.

At the very least, IMHO, one must first purify the protein to near 
homogenity (sp.?), and only _then_ bind it back to GI-Sepharose. 

Hope this helps,

        - Dima



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