Non-specific antibody control for ChIP (View forum version)



XXy

Posted 15 October 2009 - 11:38 PM

Hello!

As far as I understand, a nonspecific antibody (Ab) control for ChIP should be raised in the same species as the specific Ab, and should have the same isotype. Taking into account these criteria, I used an unrelated Ab that had nothing to do with immunoprecipitation of modified histones, but matched the criteria for use as a nonspecific control, and my aspecific binding was perfect. Until I discovered that there was a much cheaper solution: ordering a control IgG. But now I have high aspecific binding... This means that, although the unrelated Ab and the control IgG are both suitable for use as a nonspecific Ab based upon the abovementioned criteria, they give different aspecific binding. This probably depends on the method of purification of the Ab...? So my question is: how do you determine the best control IgG for your ChIP experiments? And actually, isn't this aspecific binding control then a rather 'artificial' and not optimal control for nonspecific binding to the constant region of an Ab...?

Thanks for your feedback!

FYI:
- ChIP with anti-H3K27me3 (Upstate): rabbit polyclonal IgG, protein A purified (nice, low Ct values);
- first nonspecific Ab: anti-Lyn, rabbit polyclonal IgG, affinity purified Ab (low nonspecific binding, high Ct values);
- second nonspecific Ab: Rabbit-Control IgG-ChIP grade (Abcam): rabbit polyclonal IgG, purified with affinity chromatography using mouse IgG-agarose (high nonspecific binding, low Ct values)

Dr Teeth

Posted 16 October 2009 - 05:15 AM

Hello!

As far as I understand, a nonspecific antibody (Ab) control for ChIP should be raised in the same species as the specific Ab, and should have the same isotype. Taking into account these criteria, I used an unrelated Ab that had nothing to do with immunoprecipitation of modified histones, but matched the criteria for use as a nonspecific control, and my aspecific binding was perfect. Until I discovered that there was a much cheaper solution: ordering a control IgG. But now I have high aspecific binding... This means that, although the unrelated Ab and the control IgG are both suitable for use as a nonspecific Ab based upon the abovementioned criteria, they give different aspecific binding. This probably depends on the method of purification of the Ab...? So my question is: how do you determine the best control IgG for your ChIP experiments? And actually, isn't this aspecific binding control then a rather 'artificial' and not optimal control for nonspecific binding to the constant region of an Ab...?

Thanks for your feedback!

FYI:
- ChIP with anti-H3K27me3 (Upstate): rabbit polyclonal IgG, protein A purified (nice, low Ct values);
- first nonspecific Ab: anti-Lyn, rabbit polyclonal IgG, affinity purified Ab (low nonspecific binding, high Ct values);
- second nonspecific Ab: Rabbit-Control IgG-ChIP grade (Abcam): rabbit polyclonal IgG, purified with affinity chromatography using mouse IgG-agarose (high nonspecific binding, low Ct values)



Mouse or rabbit control IgGs are often pools of IgGs, not a single IgG.