title: High-throughput electrophoretic mobility shift assays for quantitative analysis of molecular binding reactions.
abstract: We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch-ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets.
authors.affiliation: Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley , Berkeley, California 94720, United States.
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Pan
authors.forename: Todd A
authors.initials: TA
authors.lastname: Duncombe
authors.forename: Colleen A
authors.initials: CA
authors.lastname: Kellenberger
authors.forename: Ming C
authors.initials: MC
authors.lastname: Hammond
authors.forename: Amy E
authors.initials: AE
authors.lastname: Herr
mesh_terms: D001665:Binding Sites; D003198:Computer Simulation; D024202:Electrophoretic Mobility Shift Assay; D044085:Microfluidics / Q000379:methods*