I am trying to do CO-IP and I used two kits: Pierce™ Co-Immunoprecipitation Kit and Pierce™ Immunoprecipitation Kit. In both cases I don't have my target band (around 100 kDa) and I only have a 50 kDa that I assume that is the heavy chain from the antibody. I don't know what to do. I have a control of my lysate and I do get a band at the right size.
So it sounds like the antibody is working but the Co-IP assay is unfortunately not working. I am not familiar with those kits in particular but a couple things you might want to try is seeing if there is any kind of issue eluting from the beads. You could have you protein bound to the beads and not being eluted. Could try stronger elution conditions. You could also use a positive control and do a Co-IP of things that you know are more highly expressed such that you can then see if it may be an issue simply with a low recovery.
Joshua Beytebiere thank you! What elution buffer would you suggest? i use that elution bufffer the comes with the kit
You can use the same buffer to start with at least and try a longer elution time. You can also try the following:
50mM TRIS-HCL pH8.0
10mM EDTA pH8.0
1% SDS
1mM DTT (add DTT just prior to use)
Well let's get more data in the question and better help. Did you load lanes with cell lysate taken both before and after your attempt to co-IP?
Now do you see both the target of the pull down antibody and the protein you are hoping to Co-IP in the pre-immunoprecipitation lysate when you develop blots accordingly for western?
Does the target of the pull down antibody diminish at at between the pre-IP lysate and the post-IP lysate?
Now most importantly, do you see the protein that you are attempting to co-IP in the pre-IP lysate?
I have had similar struggle as likely most who do this co-IP. Proving that your co-IP ultimate target is detectable in your sample before IP and proving that the initial IP works are necessary internal controls, even before "co" part of the co-IP is relevant.
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