We are trying to design an experiment investigating chromatin co-localization of two DNA binding proteins in the system where both endogenous genes are knock-out and re-expressed with attached tags. Most of published Re-ChIP experiments that I came across utilize one anti-tag antibody and second protein-specific. That makes me wonder if there are some technical or conceptual disadvantages of using two tags in this type of experiments. Thank you in advance for all your input.
You should be fine I would agree with Wael R Abdel-Fattah that you will want two different types of antibodies for the different IP's. I would also add that you will be dealing with a very small amount of material likely since you IP the material twice and will only get areas that are bound by both proteins so I would remember that you will have very little DNA coming out of the IP.
Joshua's point about the second IP is a good one, there is way less material at this stage, so be sure to massively upscale your first IP. Using at least one tag is almost essential, because how you elute from the first IP is actually a major challenge. If you use detergent, the unfolded input to the second IP will be much more sticky, compromising the specificity (in some cases making it essentially a non-specific precipitation). So using a mild-elutable tag for your first IP can really circumvent this problem-- FLAG is great for this, as you can get pretty good elution with 3xFLAG peptide, or some sort of specific protease cleavage step to release protein from the resin. Be sure to have controls for places you know both exist, one of the two epitopes will exist, and where they will be absent, so that you can interpret your otherwise very challenging to normalize data. Also, do a tube change with every wash, to reduce background.
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