RNA-binding protein purification and identification (RaPID) was performed essentially as described by Slobodin and Gerst, 2010 with a few modifications. In order to block endogenous biotinylated moieties, the protein aliquot taken for pull-down was incubated with 10 mg of free avidin (Sigma) per 1 mg of protein input at 4°C for 1 hr with constant rotation. In parallel, streptavidin magnetic beads (New England Biolabs) were washed five times with 1 ml of lysis buffer supplemented with EDTA 5 mM. Pull-down was then performed by adding the indicated amount (see figure legends) of avidin-blocked total cell lysate to the beads, followed by incubation at 4°C for 3 hr with constant rotation. Beads with captured mRNPs were washed three times with lysis buffer and three times with washing buffer (25 mM Tris-HCl, pH 7.5, 300 mM KCl, 0.5% NP40, 1 mM DTT, 5 mM EDTA, and 40 U/ml RNAse inhibitor) (New England Biolabs) at 4°C for 5 min to remove non-specifically associated proteins. For elution of the cross-linked RNP complexes, 250 µl of washing buffer supplemented with 6 mM free biotin (Sigma) was added to the beads, followed by 1 hr of incubation at 4°C with rotation. To reverse the cross-link for RNA analysis, samples were incubated at 70°C for 1 hr with cross-link reversal buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM DTT, and 1% SDS).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.