A Platform to Enable the Pharmacological Profiling of Small Molecules in Gel-Based Electrophoretic Mobility Shift Assays.
We describe a polyacrylamide gel casting cassette that overcomes limitations of commercially available gel electrophoresis equipment. This apparatus molds a single polyacrylamide gel that can evaluate more than 200 samples in parallel, is loaded with a multichannel pipettor, and is flexible with respect to composition of the separating matrix. We demonstrate its use to characterize inhibitors of enzymes that modify protein and nucleic acid substrates. Throughputs of greater than 1000 samples per day were achieved when this system was paired with a quantitative laser-based imaging system, yielding data of remarkable quality.
1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.|Primary Pharmacology Group, Pharmacokinetics, Dynamics & Metabolism-New Chemical Entities, Pfizer, Groton, CT, USA.
Timothy L
TL
Foley
1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
Dorjbal
D
Dorjsuren
1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.|Assay Development and Drug Repurposing Core Facility, Department of Pharmacology & Toxicology, Michigan State University, East Lansing, MI, USA.
Thomas S
TS
Dexheimer
2 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.
Michael D
MD
Burkart
3 Luxon Engineering, LLC, San Diego, CA, USA.
William C
WC
Wight
1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
Anton
A
Simeonov
D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D011485:Protein Binding; D054852:Small Molecule Libraries / Q000032:analysis / Q000737:chemistry*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries
electrophoretic mobility shift assay (EMSA); electrophoretic techniques; enzymatic assays and analyses; high-throughput screening (HTS); polyacrylamide gel electrophoresis (PAGE); small-molecule testing