Does anyone know of someone doing CLIP or RIP in c. elegans? I’m looking for a protocol.
I sent the protocol for RIP to your email.
Basically, I have a 3xFLAG labeled protein, sonicate animals, immunoprecipitate by anti-FLAG, wash, elute, precipitate. It is very straigforward.
I am also interested in CLIP and RIP protocols. Can anyone please email me these protocols. Thanks!
you don’t have an email address available.
Tried your protocol, the RNA looks great. Only problem is it is in every step. I don’t have a difference between with and without antibody. Any ideas? I thought maybe the RNA was sticking to the tubes.
We have used a ‘CLIP’ protocol called mRNA-tagging to immunoprecipitate 3xFLAG-tagged poly-A binding protein crosslinked to mRNA. In our optimization of Peter Roy and Stuart Kim’s original protocol we determined that RNA sticks non-specifically to sepharose beads. To get around this obstacle in our microarray experiments we compare two IPs against each other (e.g. wt and mutant or N2 and 3xFLAG::PAB transgenic) to identify what genes are enriched over background and between the two conditions.
For the original protocol see Roy et al 2002. For our updated protocol see Von Stetina, Watson et al 2007. Hope this helps!
In our RIP experiments, we always compare WT and mutants, or treatment vs non-treatment. We can get several hundred enrichment of our target RNA comparing to controls. However, the unspecific RNA is alway not changed.