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Protein and lipid kinases are involved in a wide variety of pathological conditions. Accordingly, kinase biology constitutes a critical field for drug discovery research.  Bioluminescent assays that measure the transfer of phosphate from ATP to cellular substrates enable rapid screening of inhibitors, selectivity profiling and pathway analysis in high-throughput formats.

Kinase Biochemical Activity Assays

Count on minimal interference when screening your compound library with luminescent, ATP/ADP-based kinase assays. These sensitive and reliable assays are easily scaled to meet your throughput needs.


Schematic Overview of the ADP-Glo™ Kinase Assay

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Detect Activity of a Range of Kinases

The ADP-Glo™ Kinase assay has a high dynamic range and produces a strong signal at low ATP to ADP conversion, making it well suited for screening low activity kinases such as growth factor receptor tyrosine kinases. The assay can be used at ATP concentrations up to 1mM, important for kinases with high Km values for ATP.


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The ADP-Glo™ Assay is a universal assay that works with any kinase and substrate. Representative examples of kinase activity curves are shown here.

 


Kinase Enzyme Systems

Easily screen and profile kinase inhibitors with Kinase Enzyme Systems.

  • 174 available, 200+ coming soon!
  • Each Kinase Enzyme System includes pre-qualified enzyme, substrate and reaction buffer optimized for use with ADP-Glo™ Assays
  • All Kinase Enzyme Systems have Application Notes available so you can get started quickly
  • Kinases are available in strip format for easier profiling (see Kinase Selectivity Profiling Systems)

The Kinase Enzyme Systems Span the Breadth of the Human Kinome

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Kinase Selectivity Profiling Systems

The Kinase Selectivity Profiling Systems are optimized for fast and simple kinase profiling reactions (Figure below) and include kinase and substrate pairs grouped in single kinase families or as a general panel of kinases representative of the human kinome for a broad kinase profile. These systems rely on the proven ADP-Glo™ Kinase Assay technology to make kinase profiling fast and simple. Systems can also be customized.

  • Fast turnaround time in a matter of hours
  • Flexible kinase inhibitory profiling
  • Stable luminescent signal allows batch plate processing

Schematic Overview of the Kinase Selectivity Profiling System

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Concentrated kinases and substrates/cofactor stocks are first diluted and then combined with test compound in a 384-well plate. After a 1-hour incubation, the ADP-Glo™ Assay is performed. The resulting luminescent signal is proportional to ADP concentration and correlates with kinase activity. 


Kinase Cellular Assays

NanoBRET™ Target Engagement Intracellular Kinase Assays

The NanoBRET™ Target Engagement (TE) Intracellular Kinase Assay is based on the NanoBRET™ System, an energy transfer technique designed to measure molecular proximity in living cells. The NanoBRET™ TE Assay measures the apparent affinity of test compounds by competitive displacement of the NanoBRET™ tracer, reversibly bound to a NanoLuc® luciferase-kinase fusions in cells. 

  • Currently >275 assays available, with more coming soon!
  • Quantitatively measure test compound affinity and fractional occupancy  for kinases in live cells for more physiologically relevant information.
  • These assays measure the interaction between full-length kinases and test compounds.
  • Ready-to-use,  kinase-specific assays offer a simple workflow and high-throughput compatibility
  • Detect multiple kinase inhibitor types, such as inhibitors with different binding modes (including type I, II, allosteric and covalent) 
  • Measure the durability of test compound binding to kinase (residence time) using kinetic analysis 

Applications of NanoBRET™ Target Engagement Intracellular Kinase Assays

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The assays can measure test compound affinity under equilibrium conditions (upper panel), or residence time under non-equilibrium conditions (lower panel).


Schematic Overview of the NanoBRET™ TE Kinase Assay Workflow

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NanoLuc®-Abl Kinase (Live Cells)

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Abl Kinase Residence Time

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Examining both cellular affinity and residence time in live cells can provide a more complete picture of a compound engaging a kinase. This example uses the NanoBRET™ TE ABL1  kinase assay and several drugs targeting chronic myelogenous leukemia (CML). The first-generation CML drug Imatinib shows weaker binding and shorter residence time at ABL1, compared to second-generation (Dasatinib) and third-generation (Ponatinib) drugs. Ponatinib displays similar affinity to Dasatinib but has much longer residence time.


Protein Phosphorylation and Pathway Analysis


These sensitive luminescent assays use NanoBiT® secondary antibody detection to examine phosphorylation and study inhibitors that target specific nodes of the major kinase signaling pathways.

  • Less interference from chemical compounds; only a luminometer is required
  • Homogeneous “add and read” format amenable to high-throughput screening 
  • Endogenous substrates eliminate the need for cell engineering and assays are compatible with any cell type, including primary cells
  • Sensitive detection allows for assays to be performed on a low number of cells
  • No immunoprecipitation step needed to detect phosphorylated protein
  • “Do it yourself” assay can be adapted to any pathways or targets of interest

NanoBiT® Immunoassays for Detecting Protein Phosphorylation

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An overview of NanoBiT® Immunoassays and the workflow. The assay is rapid and homogeneous, with an easy “add and read” format.


 

Interested in more information about protein phosphorylation and pathway analysis?

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Kinase Biology Resources

Automating the Kinase Profiling Systems

Learn how to use Promega kinase assays for rapid screening of compound libraries.

Watch Video

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ADP-Glo: A Luminescent ADP Detection Assay for Kinases

In vitro studies of the mode of action of kinase inhibitors, and drug resistance mechanisms of mutated kinases.

Download Poster

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Application of BRET Technology to Quantitatively Determine Kinase Inhibitor Potency in Live Cells

Discover how a collaboration between Promega and Reaction Biology Corporation has enabled conversion of kinase target engagement assays into a service compatible with high-throughput cell-based profiling.

View Webinar