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Protocol to measure protein-RNA binding using double filter-binding assays followed by phosphorimaging or
Joel Vega-Badillo1, Phillip D Zamore2, Karina Jouravleva1
1RNA Therapeutics Institute, University of Massachusetts Chan Medical School, 368 Plantation Street, Worcester, MA 01605, USA.
STAR Protocols
|June 4, 2023
Summary
This study details a method to measure microRNA-loaded Argonaute2 protein binding affinity (KD) using double filter binding. The protocol is adaptable for various RNA- and DNA-binding proteins.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Binding affinity quantifies molecular interaction strength, often reported as the equilibrium dissociation constant (KD).
- Accurate measurement of KD is crucial for understanding protein-RNA interactions in biological systems.
Purpose of the Study:
- To present a detailed protocol for measuring the binding affinity (KD) of mammalian microRNA-loaded Argonaute2 protein.
- To provide a method applicable to other RNA- and DNA-binding proteins.
Main Methods:
- The double filter binding assay was employed to measure binding affinity.
- Key steps include radiolabeling target RNA, determining binding-competent protein concentration, setting up binding reactions, separating bound and unbound RNA, library preparation for Illumina sequencing, and data analysis.
Main Results:
- A robust protocol for measuring KD of microRNA-loaded Argonaute2 protein was established.
- The protocol's steps are clearly delineated for reproducibility.
Conclusions:
- The presented double filter binding protocol offers a reliable method for quantifying binding affinity of microRNA-loaded Argonaute2.
- This methodology is versatile and can be adapted for diverse RNA- and DNA-binding proteins, facilitating broader research in molecular interactions.
