Co-IP, nuclear proteins - buffers for lysis? - Who else does co-ip on nuclear proteins? (Mar/23/2006 )
OK, so now I have tried to set up my co-ip to check some stable protein-protein interactions. I have been using a buffer with NP-40 and Triton-X. It seems that I get some interactions, but not with those proteins I wanted. And I know they are there.
My washing buffer isn't of high stringency, so I thought it's the fault of lysis buffer (again). My protein complex of interest is localized in nucleus.
Who else of you does co-ip of nuclear complexes? Which kind of lysis buffer and detergents do you use? I can't really buy a kit, and I start being desperate. I'll be grateful for any hints :/
-Telomerase-
QUOTE (Telomerase @ Mar 23 2006, 03:28 AM)
OK, so now I have tried to set up my co-ip to check some stable protein-protein interactions. I have been using a buffer with NP-40 and Triton-X. It seems that I get some interactions, but not with those proteins I wanted. And I know they are there.
My washing buffer isn't of high stringency, so I thought it's the fault of lysis buffer (again). My protein complex of interest is localized in nucleus.
Who else of you does co-ip of nuclear complexes? Which kind of lysis buffer and detergents do you use? I can't really buy a kit, and I start being desperate. I'll be grateful for any hints :/
My washing buffer isn't of high stringency, so I thought it's the fault of lysis buffer (again). My protein complex of interest is localized in nucleus.
Who else of you does co-ip of nuclear complexes? Which kind of lysis buffer and detergents do you use? I can't really buy a kit, and I start being desperate. I'll be grateful for any hints :/
yes me too in deep trouble with this. everytime i do a coip for nuclear proteins i get a good smear in the region of my interest in the western blot. my collegue who repeated the same protocol except the interaction is cytoplasmic had good results and neat bands...ready for publication...
anybody experienced with nuclear coip?
thx
-rajgene-