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

Matthew B Robers; Jennifer M Wilkinson; James D Vasta; Lena M Berger; Benedict-Tilman Berger; Stefan Knapp

doi:10.1016/j.xpro.2021.100822

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

STAR Protoc. 2021 Sep 15;2(4):100822. doi: 10.1016/j.xpro.2021.100822. eCollection 2021 Dec 17.

Authors

Matthew B Robers¹, Jennifer M Wilkinson¹, James D Vasta¹, Lena M Berger², Benedict-Tilman Berger², Stefan Knapp²

Affiliations

¹ Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53719, USA.
² Structural Genomics Consortium, Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences, Max-von-Laue-Straße 15, 60438 Frankfurt am Main, Germany; Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.

Abstract

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).

Keywords: biotechnology and bioengineering; cell biology; cell-based assays; high-throughput screening; molecular biology; molecular/chemical probes.

MeSH terms

Biological Assay*
Luciferases
Phosphotransferases*

Substances

Luciferases
nanoluc
Phosphotransferases