We have a small DNA promoter motif that is essential for expression of our favorite gene. We would like to find the transcription factor target(s) that binds to it. I am unsure if use of nuclear extracts/EMSA/MS will be too insensitive an approach to do this - or if I need to go to alternate assays. If anyone has successfully employed such an approach I would much appreciate discussing protocol & pitfalls. Thanks.
Using a DNA sequence to isolate binding proteins is possible but not easy. You must prepare lots of nuclear extracts and remove non-specific DNA binding proteins, of which there are many. You can then bind the oligo to a solid support by using a biotinylated oligo and streptavidin agarose, for example. Pass the cleared extract over a column and elute with increasing salt concentration. Samples are then sent for mass spec.
However, you might want to consider a genetic approach, whereby DS-RNA against various transcription factors are injected into C.elegans and expression of your gene is monitored. This might be faster in the long run, especially if you can narrow down the transcription factors to a handful of candidates based on literature and expression patterns.
Hi Shane, yes we have done this but it also required some purification before running the EMSA. The procedure is described in an older paper:
Expression of Pax2 in the intermediate mesoderm is regulated by YY1. Patel SR, Dressler GR. Dev Biol. 2004 Mar 15;267(2):505-16.
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