Ribonucleoprotein immunoprecipitation protocol

Ribonucleoprotein Immunoprecipitation Protocol Ribonucleoprotein (RNP) is a combination of RNA and RNA-binding proteins that drives the regulation of post-transcriptional gene expression. A detailed understanding of RNPs provides valuable information for not only the knowledge of particular pathways, but also development of novel compounds representing potential therapeutic targets and biomarkers.

RNA immunoprecipitation (RIP) is an antibody-based technique that allows the immunoprecipitation and isolation of transcripts (mRNAs or microRNAs) and protein components of RNP complexes from cell extracts. RNAs can be extracted from the purified RNA-protein complexes and further detected by various molecular biology tools including cDNA sequencing, microarrays or real-time PCR. This protocol provides a detailed procedure to obtain high-qualify RNA for a successful RIP assay.

Reagents: Polysome lysis buffer: 150 mM KCl, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.5% NP-40, 0.5 mM DTT, with 100 U/ml RNAase inhibitor and 1Ă— protease inhibitor cocktail.

NT2 buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40, with 1Ă— protease inhibitor cocktail.

NT2-Ab coupling buffer: NT2 buffer supplemented with 5% BSA. Nuclease-free PBS: pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4. https://www.creative-diagnostics.com/ribonucleoprotein-immunoprecipitation-rip-p rotocol.htm