I want to identify the unknown RNA binding protein(RBP) in few mRNAs of interest. I am looking for a reliable immune-precipitation method (or any other possible method) to find the unknown RBP possibly attached to known mRNA.
Hi
I'd suggest doing something like this:
1. Harvest cells (optional treatment of cells with formaldehyde to cross-link in vivo protein-RNA complexes)
2. Isolate nuclei and lyse nuclear pellets
3. Shear chromatin
4. Immunoprecipitate the RNA binding protein (RBP) of interest together with the bound RNA
5. Wash off unbound material
6. Purify RNA that is bound to immunoprecipitated RBP
7. Reverse transcribe RNA to cDNA and analyze by qPCR, microarray or sequencing
For a more in-depth protocol, I'd suggest looking up RNA immunoprecipitation (RIP) protocols, of which you can find a lot.
Hope I could help
Art
Hi Art Schuermans
Thank You for the answer. But, my real problem is I am trying to find RBP bound to mRNA so I cant use antibody again RBP protein (since I dont know the RBP attached ) for IP.
Are you expressing your cDNA-of-interest in your own vector system? If so, you could fuse them to MS2 hairpin sequences and then IP with MS2 binding proteins. This is an established technique called MS2-TRAP. I haven't done it myself but a postdoc in our lab used to do it a lot a few years ago, seemed to work pretty well.
Two options.
1. RAP (RNA antisense pulldown): You crosslink the cells with UV and incubate the extract with biotinylated antisense probes. Pulldown the bound complexes, extract the bound proteins and send to mass-spec.
2. Add biotinylated, in-vitro transcribed, RNA, pulldown bound proteins and send to mass spec.
The second option is easier, the first option has the advantage of pulling native complexes formed in the cells.
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