RNA immunoprecipitation...HELP! - (Apr/21/2010 )

Hi all,

I really hope someone can help me-I've been studying a really interesting RNA binding gene whose targets are not particularly well established.

I would really like to do a RIP-SEQ on it however my experience of Protein work/IP is extremely limited and unfortunately no one in my lab is much help either!. For that reason i was planning on using a kit but i would like some advice even if it is just to say I'm wasting my time on too complicated an experiment for someone with no experience.

I'm mostly confused about the best ways to check if the efficiency of the IP and that IP has worked correctly given that I don't know the binding targets.

if anyone has an advice-I would really appreciate it.
thanks!!

quinner on Wed Apr 21 15:01:41 2010 said:

Hi all,

I really hope someone can help me-I've been studying a really interesting RNA binding gene whose targets are not particularly well established.

I would really like to do a RIP-SEQ on it however my experience of Protein work/IP is extremely limited and unfortunately no one in my lab is much help either!. For that reason i was planning on using a kit but i would like some advice even if it is just to say I'm wasting my time on too complicated an experiment for someone with no experience.

I'm mostly confused about the best ways to check if the efficiency of the IP and that IP has worked correctly given that I don't know the binding targets.

if anyone has an advice-I would really appreciate it.
thanks!!

avalon on Tue May 10 20:33:27 2011 said:

quinner on Wed Apr 21 15:01:41 2010 said:

Hi all,

I really hope someone can help me-I've been studying a really interesting RNA binding gene whose targets are not particularly well established.

I would really like to do a RIP-SEQ on it however my experience of Protein work/IP is extremely limited and unfortunately no one in my lab is much help either!. For that reason i was planning on using a kit but i would like some advice even if it is just to say I'm wasting my time on too complicated an experiment for someone with no experience.

I'm mostly confused about the best ways to check if the efficiency of the IP and that IP has worked correctly given that I don't know the binding targets.

if anyone has an advice-I would really appreciate it.
thanks!!

Hi Quinner - I am in a similar position! Did you manage to figure out how to do this? Any help greatly appreciated!

Did any of you guys have problems with non specific RNA binding to the agarose beads used in the IP? Im having a lot of issues with this that I cant seem to resolve

Hi all,

I am trying to do a similar experiment and I do not know the targets of my protein. The amount of RNA that I get after IP is pretty low (~200-300ng). How can I be sure that it is RNA indeed? On bioanalyzer (RNA nano kit), I do not see discrete bands, though it gives the concentration.