Protocol to measure protein-RNA binding using double filter-binding assays followed by phosphorimaging or high-throughput sequencing.
STAR Protoc
; 4(2): 102336, 2023 Jun 03.
Article
en En
| MEDLINE
| ID: mdl-37270783
ABSTRACT
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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STAR Protoc
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2023
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Article