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  • An Optimized Immunoprecipitation Protocol for Assessing Protein-RNA Interactions In Vitro.

An Optimized Immunoprecipitation Protocol for Assessing Protein-RNA Interactions In Vitro.

STAR protocols (2020-10-01)
Alejandro Fuentes-Iglesias, Vera Garcia-Outeiral, Jose Angel Pardavila, Jianlong Wang, Miguel Fidalgo, Diana Guallar
ABSTRACT

RNA-binding proteins are key regulators of cell identity and function, which underscores the need for unbiased and versatile protocols to identify and characterize novel protein-RNA interactions. Here, we describe a simple and cost-effective in vitro RNA immunoprecipitation (iv-RIP) method to assess the direct or indirect RNA-binding ability of any protein of interest. The versatility of this method relies on the adaptability of the immunoprecipitation conditions and the choice of the RNA, which exponentially broadens the range of potential applications. For complete details on the use and execution of this protocol, please refer to Guallar et al. (2020).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Millipore
Benzonase® Nuclease, ≥250 units/μL, ≥90% (SDS-PAGE), recombinant, expressed in E. coli, buffered aqueous glycerol solution
Supelco
Bradford Reagent, for 0.1-1.4 mg/ml protein
Sigma-Aldrich
ANTI-FLAG® M2 antibody, Mouse monoclonal, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, ACS reagent, ≥99.5%
Sigma-Aldrich
Gelatin from bovine skin, Type B, powder, BioReagent, suitable for cell culture
Roche
Glycogen, from mussels