We are interested at looking for a protein complex that may bind DNA. The protein of interest doesn't appear to directly bind DNA (doesn't have a predicted DNA binding sequence) but localizes to the nucleus and apprears to be bound to DNA by 3D-SIM microscopy. How would we go about testing if this protein bound to DNA as part of a multi-protein complex? Would ChiP-seq be able to show that or is some other method optimal to detect a protein complex bound to DNA?
Hi John,
If a protein of interest is not bound directly to DNA, ChIP still can be used but you have to play with fixation to be sure that you catch the whole complex. A dual fixation using FA together with another fixators, like DSG, has been used successfully.
HI John,
If you know the components of your protein complex and DNA sequence, I would suggest you can do gel-shift assay (also known as EMSA), better if you do DNase I Footprinting with DNA being chemically bound to fluorophore molecules. Footprinting followed by DNA sequencing will precisely tell you what DNA sequence it binds to. I am assuming you have some idea of what DNA it binds to based on the type of the protein. Now to get a quantitative measure as to how strongly the protein binds and in which order, you can try ITC or Fluorescence Anisotropy where you add protein in different order to DNA in solution to understand how they form an active complex before being DNA bound. This will also give you an insight into the mechanism of protein-DNA binding, perhaps if you are the first one to study this system.
I have attached a link of my paper that describes all these methods. Let me know if you have any more question.
Goof Luck!
Sugata
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