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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
摘要

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).

材料
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品牌
产品描述

Millipore
Benzonase®核酸酶, ≥250 units/μL, ≥90% (SDS-PAGE), recombinant, expressed in E. coli, buffered aqueous glycerol solution
Sigma-Aldrich
纯乙醇, 200 proof, ACS reagent, ≥99.5%
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
单克隆抗-FLAG® M2 小鼠抗, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
Supelco
Bradford试剂, for 0.1-1.4 mg/ml protein
Roche
糖原, from mussels
Sigma-Aldrich
明胶 来源于牛皮, Type B, powder, BioReagent, suitable for cell culture