A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells

STAR Protocols

Kelly M. Gatfield¹  Daniel A. Thomas²  John P. Evans³  Charles S. Lay⁴  Peter D. Craggs⁵  Emma J. Jones⁵ 

[1] Arctoris, Oxford OX14 4SA, UK;Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, UK;Medicine Design, Medicinal Science and Technology, GlaxoSmithKline, Stevenage SG1 2NY, UK;Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK;Medicine Design, Medicinal Science and Technology, GlaxoSmithKline, Stevenage SG1 2NY, UK;

关键词: Cell Biology;    Cell-based Assays;    High Throughput Screening;    Molecular/Chemical Probes;   

DOI  :  

来源: DOAJ

【 摘 要 】

Summary: The Kinetic Intra-Cellular Assay (KICA) is a recombinant cell-based technique that utilizes NanoBRET technology. KICA enables the measurement of intracellular binding kinetics. This protocol describes steps for cellular transfection and expression, followed by addition of a target specific fluorophore conjugated probe and a range of concentrations of competitor compounds, followed by the measurement of BRET in a 384 well format. Fitting the BRET data allows measurement of forward and reverse binding rates and the determination of KD.For complete details on the use and execution of this profile, please refer to Lay et al. (2021).

【 授权许可】

Unknown