1 reply to this topic

[research_freak]

Hey Guys,

I noticed that most of my GST-tagged protein comes into the soluble fraction during large-scale expression. When I tried to purify it on glutathione beads, most of my protein remains bound to the beads but fails to elute. Is there a way I can elute all of my protein?

Any advice is greatly appreciated.

[RMcKenney]

research_freak, on Apr 9 2010, 06:07 AM, said:

Hey Guys,

I noticed that most of my GST-tagged protein comes into the soluble fraction during large-scale expression. When I tried to purify it on glutathione beads, most of my protein remains bound to the beads but fails to elute. Is there a way I can elute all of my protein?

Any advice is greatly appreciated.

I've had this problem with a stubborn protein.  I believe the protein gets aggregated on the column.  Interestingly, I inserted an enzyme cleavage site (Prescission enzyme) in between the GST and my protein of interest.  When I cut the GST off my protein, the protein is easily eluted off the column!  Remember that GST is a dimer and may be forcing your protein of interest into conformations that may make it "unhappy."

Edited by RMcKenney, 14 April 2010 - 04:21 AM.