Affiliation:
1. Sanofi, Integrated Drug Discovery Industriepark Hoechst Frankfurt am Main Germany
Abstract
AbstractThis article presents detailed descriptions of procedures and troubleshooting tips for solid‐supported membrane (SSM)‐based electrophysiology assays (SURFE²R) to measure electrogenic solute carrier transporter proteins (SLCs) and assess the effects of compounds that modulate their activity. SURFE²R allows the use of the standard 96‐well format, making it an ideal platform for tertiary assays in a drug‐discovery campaign. The assays are performed with cell‐line‐derived membrane fractions or proteoliposomes containing the transporter of interest. Three main protocols are described for the isolation of membrane fractions from cell culture and the generation of proteoliposomes containing the transporter of interest. Additionally, detailed protocols for SURFE²R single concentration and dose‐response experiments are included to measure the potencies of test compounds in stimulating or inhibiting transporter function (EC50 or IC50 values, respectively) and kinetic functional assays to calculate apparent affinity (kM) and maximal velocity (Vmax) of substrate uptake. © 2023 Sanofi. Current Protocols published by Wiley Periodicals LLC.This article was corrected on 23 March 2023. See the end of the full text for details.PROTOCOL GROUP 1: Sample preparation for SSM‐based electrophysiology assaysSupport Protocol 1: Production of cell batchesSupport Protocol 2: Simple isolation of cell membranesAlternate Protocol 1: Isolation of cell membranes with sucrose gradient pre‐purificationSupport Protocol 3: Production and isolation of liposomesSupport Protocol 4: Preparation of sensor with isolated cell membranesAlternate Protocol 2: Preparation of sensor with isolated proteoliposomesPROTOCOL GROUP 2: Determination of assay parameters for SSM‐based electrophysiology assaySupport Protocol 5: Assay with stable bufferAlternate Protocol 3: Assay with ion gradientSupport Protocol 6: Determination of membrane/liposome concentrationSupport Protocol 7: Determination of substrate dependency kMPROTOCOL GROUP 3: Determination of advanced assay parameters for SSM‐based electrophysiology assaysSupport Protocol 8: Assessment of ion concentration dependencySupport Protocol 9: Assessment of pH dependencySupport Protocol 10: Assessment of DMSO dependencySupport Protocol 11: Assessment of signal stability with multiple activationsPROTOCOL GROUP 4: Compound testing through SSM‐based electrophysiology assays using SURFE²R apparatusSupport Protocol 12: Assessment of signal specificity of a published inhibitor or unknown compound(s)Support Protocol 13: Compound wash‐outSupport Protocol 14: Statistical analysis
Subject
Medical Laboratory Technology,Health Informatics,General Pharmacology, Toxicology and Pharmaceutics,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Neuroscience
Cited by
1 articles.
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