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

Charles S Lay; Daniel A Thomas; John P Evans; Emma J Jones; Kelly M Gatfield; Peter D Craggs

doi:10.1016/j.xpro.2021.101078

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

STAR Protoc. 2022 Jan 10;3(1):101078. doi: 10.1016/j.xpro.2021.101078. eCollection 2022 Mar 18.

Authors

Charles S Lay^{1

2

3}, Daniel A Thomas^{3

4}, John P Evans³, Emma J Jones⁵, Kelly M Gatfield³, Peter D Craggs^{3

6}

Affiliations

¹ Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
² Centre of Membrane Proteins and Receptors, University of Birmingham and Nottingham, The Midlands, UK.
³ Medicine Design, Medicinal Science and Technology, GlaxoSmithKline, Stevenage SG1 2NY, UK.
⁴ Arctoris, Oxford OX14 4SA, UK.
⁵ Protein and Cellular Sciences, Medicinal Science and Technology, GlaxoSmithKline, Stevenage SG1 2NY, UK.
⁶ GSK-Francis Crick Institute Linklabs, Medicinal Science and Technology, GlaxoSmithKline, Stevenage SG1 2NY, UK.

Abstract

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 K_D. For complete details on the use and execution of this profile, please refer to Lay et al. (2021).

Keywords: Cell Biology; Cell-based Assays; High Throughput Screening; Molecular/Chemical Probes.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Fluorescent Dyes*
Kinetics

Substances

Fluorescent Dyes