CoIP - (May/30/2013 )
I did IP of protein A & western blot for protein B(CoIP). I got an IP band for protein A-Protein B but when I did IP of protein B & western blot for protein A, I don't observe any IP band!.
Is this normal? It is consistent and I see the same trend in 3 experiments now.
Please help with your inputs on this.
Is the antibody for protein B suitable for IP? Is the detection of protein A normally weak - if so, IP tends to get about 10% of the input protein and co-IP will get about 10% of the interacting proteins (i.e. 1% of the input level of interacting proteins) - so perhaps below detection levels. Try using more antibody for protein B and longer exposure for the membrane.
Yes the antibody for protein B is suitable for IP, infact both the antibodies are polyclonal antibodies and are compatible with IP. I will definitely try more antibody for protein B. Thanks for your suggestion.