I was told by some to just try the gel shift anyway, and see what I get. So I followed the kits protocol exactly, and I also supershifted using an anti-histag Ab (See Gel Shift). Everything appeared to work nicely (Lanes 4-8) except the contaminating proteins bind to my probe as well as the kits probe and cause non-specific shifts (Lane 2). To make things more confusing the anti-His tag Ab caused a supershift with the kit's probe (Lane 3)?? However when I mutate the binding site in my specific probe I eliminate the large band that I think is my protein, but still see a super shift (Lanes 9 and 10)??I have attached a picture of the membrane. Does anybody have any suggestions? Should I be including blocking agents (BSA or detergents), in my incubations? Is my protein just sticky?? Why would my protein shift the kit's probe?? Very strange. Thanks, I would really appreciate any input!!!!
Edited by mdnworms, 10 June 2009 - 12:54 PM.