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CoIP from cell lysate mix - (Mar/10/2014 )

Hello everyone,

 

I wanted to ask if anyone had experience with performing a CoIP, by overexpressing the two target proteins NOT in the same cell, but in seperate cells, then lysing them and incubating them for a certain period of time before adding the antibody-bead suspension?

Thanks in advance.

Pax

-Paxton-

I have experice with coip. But never did in different cells. I guess you want to study their interaction in a certain period of time.

-Curtis-

It should work.

-memari-

It sounds like you are doing a "run of the mill" in vitro Co-IP. Why don't you quickly purify the protein that is being expressed in each transfected cell line? I guess it just depends on how quantitative you are trying to be.

-jerryshelly1-

This sounds a lot like a pull down assay.  If you are over-expressing tagged proteins (which greatly assist in pull-downs), I agree with jerryshelly1 above - you can immunopurify them first and then use the purified protein as "bait" for lysates in your assay.  The approach you've described is a quick/dirty variant of the same assay.  It might work, but it isn't very physiological.  Is there any reason you can't either purify the protein, or express both proteins in the same cells?

As a positive control, I think you'd still want to show both recombinant proteins interacting in vitro.  Co-IP's are nasty in the way they can fool you (both erroneous positive and negative results are common), so make sure there are appropriate positive and negative controls.

 

-miST32-

Thank you for your input, I will take it into consideration. The reason why we can't purify or express the protein with the other is sort of the premise of our experiment.

-Paxton-