NanoBRET Intracellular Kinase Assay
Target-specific Assays

NanoBRET Target Engagement Intracellular Kinase Assay Services

Reaction Biology is offering NanoBRET target engagement kinase assays in collaboration with Promega. The NanoBRET Intracellular Kinase Assay measures the 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 target engagement assay employs an energy transfer technique designed to measure molecular proximity in living cells. The 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 binding affinity 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 target engagement assay serves as a great tool to determine the direct interaction of the compounds binding to target kinases in cells.

  • The NanoBRET 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
  • Kinase activity testing in intact cells can be investigated with our Cellular Phosphorylation Assay

NanoBRET Assays

NanoBRET assays are compound competition assays 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

NanoBRET TE Intracellular CDK Panel Screening (Total CDKs: 20)

Reaction Biology offers the NanoBRET™ Intracellular CDK Panel Screening service, which includes 20 clinically relevant and recognized CDK targets. This is an excellent platform for assessing test compound’s binding affinity and selectivity across a diverse panel of CDK kinases in the physiological environment of intact cells, and it provides qualitative data on the test compound’s permeability, affinity, selectivity, and residence time.

  • 10-dose IC50 duplicate format
  • Performed on a regular basis and economical
  • Fast turnaround time: get your results in around 4 weeks
  • Semi-automated processing for highly reproducible data

NanoBRET Assay Details

  • Example Data
  • Example Data: Compound Residence Time Analysis
  • Reproducibility
  • Screening Details
  • Monthly Screening Schedule
Example Data

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.

Example Data: Compound Residence Time Analysis

Example study: BTK Compound Residence Time Analysis using NanoBRETTM Kinase Assay

HEK293 cells transiently expressing BTK-NanoLuc Fusion Vector were treated with DMSO vehicle, 250 nM dasatinib, or 1 μM ibrutinib for 2 hours. The cells were washed with Opti-MEM to remove unbound drug, after which kinase tracer K10 was added and NanoBRETTM measurements were recorded in kinetic mode on an Envision 2104 Multilabel Reader. Koff values for tracer association after washout of compounds are determined using the one-phase exponential association equation with Graphpad Prism program.


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.


Monthly Screening Schedule

On a monthly basis, Reaction Biology performs compound screening on the NanoBRET panel.

Please see the upcoming screening dates here.