I am working with two DNA binding proteins and they bind at the promoter region of a protein. I want to see whether one protein recruits another or they independently interact with the DNA.Is co-IP enough to check on that? I know that Co-IP can reveal whether they have physical interaction or not, but how to check any kind of recruitment relation between those two proteins?
Thanks
Hi,Swarnali,
I agree with Kannan. You can compare Co-IP before and after DNAse treatment. If your two protein bind the same promoter, than DNAse will release the partner protein. If there is a physical interaction, than you will see the partner protein in IP.
Thank you Kannan and Anastasia.
Kannan, I just wanted to ask whether I would be able to conclude about the recruitment thing from the competitive EMSA as you have suggested or I have to do some supportive experiments?
I understand what are you suggesting, Kannan and thank you for your quick response.
But I want to say that EMSA is an in-vitro assay, have we got some sort of in-vivo assay to check the binding of proteins, to relate with Co-IP result and to conclude on the hypothesis?
Thank you very much Kannan.:-)
Now, I am going to start with Co-IP, and after my success/ failure I am going to ask you more questions about downstream experiments.
Hi, following up on this discussion, I have a question regarding the DNase treatment you suggest: Which conditions are usually used for this treatment? I have done a DNase digestion test where I treated my lysate with different concentrations of DNaseI and checked the DNA amount after incubation using GelRed. All samples looked the same, so apparently the digest was not successful. I wonder if the lysis buffer I used could inactivate the DNase or the DNase concentration was not high enough.
Hi Laura,
This is an old thread so I'm sure you've resolved it. What was the composition of your lysis buffer. DNase would be inactivated/inhibited by chelators (i.e. EDTA), reducing reagents (e.g. DTT) or SDS.
Were you doing DNase treatment for better elution of chromatin-bound proteins prior to Co-IP?
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