How to identify a protein that binds a defined DNA sequence - (Mar/22/2005 )
Hello, I'm just wondering if there is a method to "fish out" proteins that bind with a defined DNA sequence.
For example, if you are interested at gene A which is expressed in a site-specific manner, and through your promoter deletion studies you know that certain region on the promoter is crucial for this site specific expression, and now you want to know what transcription factor control this....
Nowadays, we know there are methods that allow us to find protein interaction partner (e.g immunoprecipitation, yest two hybrid) or to find DNA sequence that recognized by a particular protein (e.g. DNA footprinting, chromatin immunoprecipitation), so I am very curious to know if there is a technique available to find out proteins that bind with your DNA of interest.
A yeast-1-hybrid allows you to fish for transcription factors using your chosen promoter - good luck
You can do DNA foot printing. Treat your DNA sample with the protein preparation and run it on a gel along with the untreated control. If any protein has bound to it then its mobility will be retarded.
Elute out the complex, isolate the protein and identify it by MS finerprinting.
yes, EMSA or footprinting (gel retardation assays or nuclease protection assays) will show such protein-DNA interactions.
I seem have read this somewhere: If you see a retarded band in EMSA, isolate that band and recover the protein for sequencing.