Isolation of protein crosslinked to plasmid
Posted 11 February 2010 - 06:27 AM
my name is Tobi and I have been thinking about the following problem:
I would like to transfect cells with a luciferase reporter plasmid, then crosslink promoter-bound proteins to the plasmid dna (with formaldehyde or something...), then somehow isolate the plasmid with the bound protein and finally submit the proteins to mass spec.
hoooo, now you think this guy is nuts or should have learned his molecular biology or something like that and you might be right in both ways.
However, if there is a slight chance this might work out somehow it would be really cool for my project.
Thanks everybody for thinking it over!!!
All the best,
Posted 15 February 2010 - 08:46 PM
why don't you go ahead and try it in small-scale? Formaldehyde crosslinking should be fine as it is reversible. The only thing I would be concerned about is how to get enough plasmid/protein "back" from the transfected cells...
Posted 16 February 2010 - 03:58 AM
thank you very much for your reply!
Actually, getting the plasmid out of the cells is the part I am worried about. Formaldehyde for crosslinking is a good idea, though.
Thanks a lot
Posted 16 February 2010 - 07:33 AM
You could produce some primers fused to biotin spanning the region of interest in your plasmid (only one of the primers is neccesary to couple to biotin). Amplify the region and get enough biotinilated DNA. Then you produce hugh amounts of your cell extract and incubate in vitro with your biotinilated dna. Pull down with strep- coated beads and run a gel in which you will have all the proteins that interact with your region of interest. (I used to do this protocol and is quite easy).
You just have to get enough material for the mass spec! But I think it is easier than in your way
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