- Protocol to measure protein-RNA binding using double filter-binding assays followed by phosphorimaging or high-throughput sequencing.
Protocol to measure protein-RNA binding using double filter-binding assays followed by phosphorimaging or high-throughput sequencing.
STAR protocols (2023-06-04)
Joel Vega-Badillo, Phillip D Zamore, Karina Jouravleva
PMID37270783
RESUMEN
Binding affinity quantitatively describes the strength of a molecular interaction and is reported by the equilibrium dissociation constant (KD). Here, we present a protocol to measure KD of mammalian microRNA-loaded Argonaute2 protein by double filter binding. We describe steps for radiolabeling target RNA, measuring concentration of binding-competent protein, setting up binding reactions, separating protein-bound RNA from protein-unbound RNA, preparing library for Illumina sequencing, and performing data analysis. Our protocol is easily applied to other RNA- or DNA-binding proteins. For complete details on the use and execution of this protocol, please refer to Jouravleva et al.1.
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Sigma-Aldrich
AEBSF, Hydrochloride, AEBSF, CAS 30827-99-7, is a specific irreversible inhibitor of chymotrypsin, kallikrein, plasmin, thrombin, trypsin, and related thrombolytic enzymes. A nontoxic alternative to PMSF and DFP.
Sigma-Aldrich
Leupeptin, Hemisulfate, Microbial, Leupeptin hemisulfate, CAS 103476-89-7, is a reversible inhibitor of trypsin-like proteases and cysteine proteases