GST fusion protein won't elute
dalmiabk at phibred.com
Mon Nov 21 18:07:32 EST 1994
In article <3a3m2o$hlv at network.ucsd.edu> T. S. Pillay, tpillay at ucsd.edu
>My colleague working alongside me has this problem:
>He has tried to purify the GRB2 SH2 domain but he finds that the protein
>does not elute off the beads with glutathione(GSH). I know this sounds
>weird but has anyone experienced a similar problem with this or any other
>fusion protein. He gets good induction of expression when looking at
>whole cell lysates but very poor yields with purification. When he
>takes the GSH-sepharose beads after elution with GSH and boils it in
>laemlli buffer- he finds "tons" of the fusion protein indicating that the
>fusion protein is sticking avidly to the beads and not eluting off.
> Any ideas/suggestions would be gladly appreciated.
what glutathione concentration is your colleague using? around 10 mM
should work. but 10 mM gsh would significantly reduce the pH of a
phosphate buffer. so try using something like tris. check the pH AFTER
addition of glutathione and should be around 7 to 8.
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