I am doing CoIP to determine the interaction of my protein of interest with another protein. An antibody was used against 27kD protein to pulldown the protein complex and detection was done with an antibody against 24 kD protein. I got a sharp band in an Input lane (used as a control for the WB) as well as in the Co-IP lane at 24kD. Does it mean that my proteins are making a complex? Or should I get a band at 51kDa (27+24) in the case of a complex? It might be a silly question but I got confused a bit.
Looks like your co-IP worked well. You should not get a 51KDa band as you are running a denaturing gel (SDS). And i assume using a denaturing loading buffer.
Thank U very much for quick reply. Yes I use denaturing loading buffer.
Aneta
Looks good. If you want an additional control you could warm an aliquot of your lysate with 0.5% SDS included to 65˚C to break the interaction. Then you could do your ip for the 27kDa protein-you should not detect the 27kDa protein.
This is a good control to do as it will tell you that the interaction does depend structure of the two proteins.
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