Hi,
I am trying to perform RNA binding protein immunoprecipitation here. I have been told that using UV or paraformaldehyde to crosslink RNA and RBP beforehand can raise the quantities. But is it necessary? I'm afraid that the decrosslinking itself might affect the natural interactions. Besides, I am only interested in the RNA identities, not the exact binding side. Which method should I choose? Another thing is that does anyone have an optimised recipe for the lysis and wash buffer? I've read several pieces of protocols provided by the companies. But I can't find the recipe for the buffer. Thank you so much!
Hi Guochun,
The reason for crosslink is to preserve the interactions as well as possible? I know that CLIP can detect the exact binding site down to a single nucleotide. But what if I am only interested in "WHO" binds to the RBP of interest, not "WHERE". Is it still necessary to do the crosslinking? And how to choose between UV and paraformaldehyde? Can you give me some suggestions? Thank you!
If you know the possible protein that potentially binds to the RNA element, use the antibody to ChIP it. Otherwise, you might have to do ChIP-proteomics, which could be tough to do as the pull-downed protein may be not enough to detect..
Formaldehyde would be better than UV for crosslinking as it is reversible and not damage your RNA?
I would advice UV crosslink for RNA-protein and Formaldehyde for DNA - Protein. for UV crossling. possible protocol (doi:10.1038/nprot.2014.012)
Hi Daisy,
If you are only interested, which transcripts are bound by the protein, go for RIP (RNA immunoprecipitation), followed by real-time PCR, microarray or sequencing. Use formaldehyde crosslinking to stabilize RNA-protein complexes. Try protocol from this paper (http://dx.doi.org/10.1016/j.molcel.2010.12.032) on RNA-protein interaction studies in vivo, see suppl. material.
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