what is the perfect volume for immunoprecipitation step of ChIP? - (Feb/04/2007 )

Hi,

I just want to know what the perfect volume is for immunoprecipitation step of a ChIP experiment. Basically, 1-2ug antibody for 1 million cells used for IP, but is there a volume limit for this step which may limit the efficiency of Ab binding? I have seen 1ml, 2ml, 3.5ml during this step.

Thanks.

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I think what is important is the concentration of your chromatin. I use 200ul for IP but the chromatin is relatively concentrated (I ususually add 50 to 100ul chromatin and dilute to 200). In my case I'm not worried about background so I can IP from fairly concentrated chromatin. Others may try to dilute further to try to decrease background.

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KPDE,

Thank you for your reply. I asked this question, because I never see any record about antibody concentration. I have seen the amount of antibody used by measuring the amount of cells per IP, but what about antibody concentration measured by ug per ul in the precipitation step. I recall I have read it somewhere, the total volume should not be more than 1 ml, but if the concentration are the same (ug/ul) for the antibody, the volume should not be a problem, is it right? Is anyone what the perfect ug/ul Ab is?

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I think the only thing which the antibody concentration affects is the kinetics of binding. Since nearly everyone incubates Ab with chromatin for much longer than they need to I don't think it's much of a problem (with the exception of our fast method, in which case, if you are worried about the Ab being too dilute, just incubate for longer). The real important thing is to make sure that you have enough antibody with excess left over to bind all of your protein on the chromatin. Since people are usually using 1-4ug of antibody per ChIP, I don't think this is much of a problem either.

Cheers,
Joel

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Great!, thanks.

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