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Why negtive control detect signal in co-ip? - co-ip (Sep/15/2005 )

Hello, everyone
I am now doing CO-IP. Both the specific antibody and the control IgG could detect the signal. I thought it is non-specific binding that lead to the signal in the negative control, but pre-clear could not solve this problem. I think it is not simply non-specific binding because more than three factors have the same problems (I performed IP using anti-A antibody and detected B, C and D factors by westernblot). I am completely confused! Any suggestion is welcome. Thank you.

-bluemoon819-

It could be your IgG Heavy chain from the IP (about 50kDa I think?) that you're detecting.

If you're using the same species antibody for the IP and detection you're anti-(that species)IgG-HRP will detect the ab from the IP.

This is only a problem if your proteins of interest are the same Mw. If it is a problem you could cross-link the ab to you protein G-sepharose (mentioned in some of the other post's on this forum) or use a different species antibody for the detection.

Hope this helps.

Ceri

-Ceri-

thanks Ceri:
My protein of intrested are 36kDa, 12kDa and 90kDa, none of them may superpose on 50kDa or 20kDa of IgG heavy or light chains. I could found the clear IgG chains at 50kDa and 20kDa posion.
looking for your help again,
Thanks

-bluemoon819-

Sorry for not replying earlier. Could it just be carry over from the lysate? Have you run some of your input lysate on the gel as well (say 20-30┬Ál i.e. a small percentage of the volume that when into the IP)? If the bands you're seeing in the IP aren't significantly stronger that the input lysate it implies this is carry over from the lysate and is not coming down with you IP'd protein.

Do you know if these proteins should interact? Also are you over-expressing these proteins or using "native" cells/cell lines ? Can you detect a protein that you know interacts with the IP'd protein as a positive control for you IP?

I doing some IPs now which I'm not seeing significant binding of the proteins above the input lysate (4% volume that went into the IP) which I've hoped to see binding to my protein of interest. I'm know going to blot for proteins I know should definitely interact to make sure I can detect these and obviously my IP'd protein. Then I'll be sure that my IPs have worked and there isn't an interaction. I thought there was the potential for an interaction from the literature but that's life.

Yours cynically,
Ceri

-Ceri-