Protocol to measure protein-RNA binding using double filter-binding assays followed by phosphorimaging or high-throughput sequencing

Binding affinity quantitatively describes the strength of a molecular interaction and is reported by the equilibrium dissociation constant (K_D). Here, we present a protocol to measure K_D 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.