TF affinity chromatography - (Mar/01/2005 )
Is there any feasibility in using a transcription factor (protein) in affinity chromatography to isolate DNA that it may bind in vivo?
By this I mean fixing a protein to a column and then passing DNA through the column to find the DNA that the protein might bind in vivo.
If so what is the ideal size for the DNA and does anyone know any protocols?
Thankyou for your help
Mmm, that's an interesting suggestion. I've seen it done the other way round (DNA-agarose columns to purify a TF). I've also seen columns of protein to purify interacting proteins, although I've never personally used one or the other techniques. I guess your suggestion could work, but it would be difficult and time-consuming to set up such an assay. Specifically, I'm concerned about which will be the washing conditions. And then you would have to clone and sequence the DNA fragments you purify that way; and you will need to use a negative control, to discard non specific binding (and the best control would be the same TF with the DBD mutated or deleted so that it doesn't bind to DNA). As for the lenght of the fragments of DNA I would use, I would sonicate or digest the DNA with nucleases to get to the 1-nucleosome level, or maybe instead of using DNA you cold use degenerate oligos...
Sorry, not much help, just random thoughts...