GST pull-down assay - (Jul/05/2001 )

I have a problem with my GST pulldown experiment. Both GST alone and GST-fusion protein pulldown the desired protein (thought it appears GST alone has a lower affinity). I have tried increasing the salt concentration, but that seems to stabilize the interaction. Any tricks to solve this problem?

Hi, maybe you should try washing the GST-beads with PBS including 0.1% nonidet-P-40. I have had the same problems with my pulldowns.Another possibility is to add BSA to your binding reaction to get rid of the unspecific binding to the GST.