GST fusion protein won't elute

bipin dalmia 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
writes:
>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.

bip



More information about the Methods mailing list