precipitation of nuclear proteins during GST pull down assay - (Nov/17/2006 )
I have been trying to do GST pull down assays using nuclear fractions. I have been doing overnight incubations with the GT fusion proteins. However the next day, invariably, my nuclear proteins precipitate. I thought this may have been due to the high salt present in my buffer, so I dialyze against 1X PBS for an hour and then concentrate the solution and resuspend the concentrated mix of proteins in appropriate buffer (low salt-120 mM KCL). Still no use.
Any facing similar problems? Any advice is appreciated.
HS
-h.s-
QUOTE (h.s @ Nov 17 2006, 03:15 PM)
I have been trying to do GST pull down assays using nuclear fractions. I have been doing overnight incubations with the GT fusion proteins. However the next day, invariably, my nuclear proteins precipitate. I thought this may have been due to the high salt present in my buffer, so I dialyze against 1X PBS for an hour and then concentrate the solution and resuspend the concentrated mix of proteins in appropriate buffer (low salt-120 mM KCL). Still no use.
Any facing similar problems? Any advice is appreciated.
HS
Any facing similar problems? Any advice is appreciated.
HS
Try dialysing in the presence of 1M NDSB 201 (1-(3-pyridinio) propanesulfonate. Will help in the solubilisation of sparingly soluble proteins without denaturing them.
-Paul T-