Single tracer-based protocol for broad-spectrum kinase profiling in live cells with NanoBRET

This protocol is used to profile the engagement of kinase inhibitors across nearly 200 kinases in a live-cell context. This protocol utilizes one single kinase tracer (NanoBRET(TM) Tracer K10) that operates quantitatively at four different concentrations. Minimizing the number of tracers offers a si...

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Detalles Bibliográficos
Autores principales: Robers, Matthew B., Wilkinson, Jennifer M., Vasta, James D., Berger, Lena M., Berger, Benedict-Tilman, Knapp, Stefan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8449129/
https://www.ncbi.nlm.nih.gov/pubmed/34568844
http://dx.doi.org/10.1016/j.xpro.2021.100822
Descripción
Sumario:This protocol is used to profile the engagement of kinase inhibitors across nearly 200 kinases in a live-cell context. This protocol utilizes one single kinase tracer (NanoBRET(TM) Tracer K10) that operates quantitatively at four different concentrations. Minimizing the number of tracers offers a significant workflow improvement over the previous protocol that utilized a combination of 6 tracers. Each NanoBRET(TM) kinase assay is built using commercially available plasmids and has been optimized for NanoLuc tagging orientation, diluent DNA, and tracer concentration. For complete details on the use and execution of this protocol, please refer to Vasta et al. (2018).

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