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

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 fluoropho...

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Detalles Bibliográficos
Autores principales: Lay, Charles S., Thomas, Daniel A., Evans, John P., Jones, Emma J., Gatfield, Kelly M., Craggs, Peter D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8760444/
https://www.ncbi.nlm.nih.gov/pubmed/35059653
http://dx.doi.org/10.1016/j.xpro.2021.101078
Descripción
Sumario: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).

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