Hello,
I am about to IP my protein of interest and maybe (I hope so) some small RNA would co-IP with it. Since this is my first time in this area, would anyone be so kind to suggest the best method for separating these RNAs for the further analysis (based on your own experience [good & bad])? I am thinking of trizol but not really sure.
Thank you in advance!
Most protein IP buffers will not inhibit RNAses which are released upon cell lysis. So that is likely to be a problem. However, you might want to look at papers that describe LNC-RNA or Micro RNA interactions with protein complexes and see how people have done this.
Hi Mark, I've been doing RNA IP with a set of RBPs, including AGO2. For extraction of co-immunoprecipitated RNA, I treat the beads directly with Trizol. Since the amount of RNA recovered is small, I include some carriers (glycogen, etc) in isopropanol precipitation, which is normally not necessary for total RNA prep. You should check your stringency of IP using a negative control like U6 snRNA, which is very abundant and therefore often co-purifies. As Gregory pointed out, don't forget to supplement your binding reaction with RNase inhibitors.
Thank you, Gregory and Dooyoung.
By "binding reaction" you mean the IP itself?
Make sure to make up ALL buffers used in the IP with RNase-free water, and RNase Inhibitors, and handle all materials in an RNase-free manner. You can use Trizol as Dooyoung suggested. It may also be possible to apply the immunoprecipitation eluant directly to the column of an RNA extraction kit like miRVANA or Qiagen's RNeasy kits.
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