NanoBRET Target Engagement Intracellular Kinase Assay Services

Reaction Biology is offering target engagement assays in collaboration with Promega using NanoBRET technology. The NanoBRET Target Engagement Intracellular Kinase Assay measures compound binding of select target kinases in intact cells and is one of two cell-based kinase assays we offer besides the Cellular Phosphorylation Assay.

The NanoBRET assay employs an energy transfer technique designed to measure molecular proximity in living cells. The NanoBRET Target Engagement Intracellular Kinase Assay measures the apparent affinity of test compounds by competitive displacement of the NanoBRET tracer, reversibly bound to a NanoLuc luciferase-kinase fusion construct in cells. The intracellular target engagement and selectivity are physiologically relevant and fundamental to the pharmacological mechanism of the compounds. While biochemical and biophysical assays identify the kinase inhibitors in vitro, the NanoBRET assay serves as a great tool to determine the direct interaction of the compounds to the target kinase in cells. 

  • The assay provides real-time data of  the binding affinity of a compound to kinase target including occupancy time 
  • Intact cells provide a relevant environment for the compound-kinase interaction in regards of ion and ATP concentration, pH, or co-factors. In addition, to function inside the cell, compounds must overcome the cellular membrane. 
  • Suitable for high-throughput screening
     

NanoBRET Assay Principle

The assay is a compound competition assay that relies on bioluminescence resonance energy transfer (BRET) between a luciferase-tagged kinase and a fluorescent tracer. Quantification and specificity are key attributes of the NanoBRET system. 
 

NanoBRET Kinase Assay Principle

Assay Details

Example data

NanoBRET Kinase Assay example with DDR1 kinase inhibition by Dasatinib. Shown are inhibition data with multiple doses and curve fitting for IC50 value determination.

Example study: DDR1 inhibition by an ATP-competitive tyrosine kinase inhibitor.

HEK293 cells transiently expressing NanoLuc-DDR1 fusion vector are seeded into 384-well plates and treated with the Tracer K-4 and compound for 1 hour. The BRET signal was measured on an EnVision 2104 multilabel microplate reader.

Reproducibility
NanoBRET kinase assay reproducibility. Shown is an example of DDR1 inhibition by Dasatinib on 130 samples.

Example study: Reproducibility of DDR1.

Assay window and Z’-factors of NanoBRET TE Intracellular Kinase Assays in 384-well plate format.

Assay conditions:

  • 4000 cells /well in 384-well assay plate
  • Compound treatment time: 1 hour
  • Tracer concentration: DDR1: 0.0625 mM K4
     
Screening details

Setups: IC50 value determination in 10-dose duplicate assay format. Free choice for screening of the kinase targets of your choice. New kinase target development/validation is available upon request.

Controls: We use DMSO as a negative control. In every project, testing of a standard reference compound is included in IC50 assay format as well.

Turnaround time: 2-3 weeks. Expedited scheduling and data delivery can be arranged prior to study commencement.

Report: A detailed report including assay conditions and comprehensive evaluation of data as well as raw data for each analysis will be provided.

Screening location: Malvern, PA, USA

Compound requirements: In brief, 50 to 100 µl of a 10 to 50 mM DMSO stock or 2-3 mg solid material is needed. Please refer to our FAQs for information regarding compound preparation and shipping.