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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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
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author Lay, Charles S.
Thomas, Daniel A.
Evans, John P.
Jones, Emma J.
Gatfield, Kelly M.
Craggs, Peter D.
author_facet Lay, Charles S.
Thomas, Daniel A.
Evans, John P.
Jones, Emma J.
Gatfield, Kelly M.
Craggs, Peter D.
author_sort Lay, Charles S.
collection PubMed
description 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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spelling pubmed-87604442022-01-19 A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells Lay, Charles S. Thomas, Daniel A. Evans, John P. Jones, Emma J. Gatfield, Kelly M. Craggs, Peter D. STAR Protoc Protocol 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). Elsevier 2022-01-10 /pmc/articles/PMC8760444/ /pubmed/35059653 http://dx.doi.org/10.1016/j.xpro.2021.101078 Text en © 2021 Glaxosmithkline https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Lay, Charles S.
Thomas, Daniel A.
Evans, John P.
Jones, Emma J.
Gatfield, Kelly M.
Craggs, Peter D.
A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells
title A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells
title_full A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells
title_fullStr A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells
title_full_unstemmed A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells
title_short A kinetic intra-cellular assay (KICA) to measure quantitative compound binding kinetics within living cells
title_sort kinetic intra-cellular assay (kica) to measure quantitative compound binding kinetics within living cells
topic Protocol
url 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
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