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

Abstract

Summary: 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

• cell biology
• cell-based assays
• high-throughput screening
• molecular biology
• molecular/chemical probes
• biotechnology and bioengineering