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CoIP


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#1 tulip

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Posted 30 May 2013 - 01:05 PM

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.
Thanks.

#2 bob1

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Posted 30 May 2013 - 02:08 PM

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.

#3 tulip

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Posted 31 May 2013 - 09:26 AM

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.




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