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Single tracer-based protocol for broad-spectrum kinase profiling in live cells with NanoBRET
Matthew B Robers1, Jennifer M Wilkinson1, James D Vasta1
1Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53719, USA.
STAR Protocols
|September 27, 2021
Summary
This protocol profiles kinase inhibitor engagement in live cells using a single tracer, NanoBRET™ Tracer K10. This streamlined method improves workflow efficiency for kinase inhibitor screening.
Area of Science:
- Biochemistry
- Pharmacology
- Cell Biology
Background:
- Kinase inhibitors are crucial therapeutics, but profiling their engagement across numerous kinases is complex.
- Previous methods required multiple tracers, complicating workflows and increasing costs.
Purpose of the Study:
- To present a streamlined protocol for profiling kinase inhibitor engagement in a live-cell context.
- To introduce a single-tracer NanoBRET™ assay for quantitative kinase profiling.
Main Methods:
- Utilized NanoBRET™ Tracer K10 at four concentrations for quantitative profiling.
- Developed assays using commercially available plasmids, optimizing NanoLuc tagging, diluent DNA, and tracer concentration.
- Applied the protocol to profile engagement across nearly 200 kinases.
Main Results:
- Demonstrated a simplified workflow by reducing the number of tracers from six to one.
- Achieved quantitative profiling of kinase inhibitor engagement in live cells.
- Established optimized assay conditions for NanoBRET™ kinase assays.
Conclusions:
- The single-tracer NanoBRET™ protocol significantly improves workflow efficiency for kinase inhibitor profiling.
- This method provides a robust and quantitative approach for assessing kinase inhibitor activity in a cellular environment.
- The protocol is adaptable and utilizes readily available reagents for broad applicability.
