I recently performed a Co-IP and I am having an issue with the precipitating antibody heavy chain masking my expected CoIPd protein's signal. I baited with a rabbit antibody and detected for the expected CoIP'd protein with a mouse antibody but it seems there is still cross reactivity between the anti-mouse HRP antibody and the rabbit heavy chain and my protein is the same size as the heavy chain :(. I had a hard enough time finding a good antibody in a different species....Does anyone have any advice on how to circumvent this issue? I was not expecting that kind of inter-species cross-reactivity.
You may want to probe your blot with light-chain specific secondary antibody, available with CST, Abcam, Jackson and all the other leading antibody production units.
Hey, this is a typical problem if you are looking for proteins that have the same size of the heavy or light chains of the antibody. My experience told me that it's independent of the source of the anitbody.
I can suggest you two different approaches depending on your kind of WB detection. If you are using ECL method, there is a special kit from Santa Cruz called Optima B/F/... (depending on your antibody species). It is optimized to reduce the detection of the heavy chain of your antibody. It also contains a light chain specific secondary antibody.
If you are using IR detection, there is a kit from Licor called "Quick Western Kit" where you don't need a secondary antibody. There is included a special detection reagent that is only recognizing the primary antibody and not the degraded IP antibody. It's also helpful if you have only one antibody.
Hello. You can try the CO-IP kit from Pierce where you bind your antibody covalently to the beads so then usually you do not have much heavy or light chains in your elution fraction. I use this kit regularly for CO-IPs and it works really well and the other good thing about this kit is that you can re-use the Ab-beads conjugates several times. Good luck.
Thanks for all of your answers! The pierce Co-IP kit is something I'll consider. I'm also trying this True Blot system which advertises that their secondary antibodies are optimized to detect only non-reduced FC domains so that may clean up my signal. It seems like the cheapest most direct solution for now. If it works that is.
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