Cellular Kinase Assays
Target-specific Assays

Cellular Kinase Assays

Cell-based assays are a great choice for secondary screening after initial hit finding because of their inherent nature of testing compound action in intact, living cells.

Compounds face multiple challenges when put to work in cells such as

  • they need to cross the plasma membrane when targeting intracellular kinases
  • they meet an abundance of other proteins that they may bind to than the target kinase, potentially leading to off-target effects causing adverse drug reactions
  • they are faced with the physiological pH, co-factors, ion concentrations in cells
  • they must act on the target in its tertiary structure complete will all post-translational modifications such as glycosylations

Read more about the importance of screening compounds in cell-based assays in our blog: Testing kinase inhibitors where it matters: Drug screening in intact cells.

To facilitate science-driven kinase drug discovery, Reaction Biology has created three different cellular platforms for kinase inhibitor screening in intact cells.

How do they compare?

Assay NanoBRET Target Engagement Assay Cellular Phosphorylation Assay BaF3 Cell Proliferation Assay
Readout Compound binding to the kinase Activity of the kinase Cell transformation capability of the kinase
Parameters Binding affinity, target residence times, target occupancy, binding kinetics Inhibitor potency Inhibitor potency, kinase-engaged signaling pathways, transformation capacity of the kinase
Assay type Competitive tracer displacement Substrate phosphorylation Cell survival and proliferation
Assay technology BRET: energy transfer caused by compound-kinase proximity resulting in luminescence ELISA or AlphaLISA to quantify phosphorylated substrate Cell proliferation assay via Cell Titer Glo
Detectable inhibitor types All All All
Kinase families All Protein kinases Oncogenic ‘driver’ kinases
Kinase expression Exogenous Endogenous or exogenous Exogenous
Compound incubation period 1-2 hours 1-2 hours 2-3 days


Our blog Spotlight: Cell-based kinase assay formats. describes our three assay formats in more detail. Check it out!

The BaF3 Cell Proliferation Assay is an activity assay for the investigation of the potency of inhibitors suppressing oncogenic kinases. It is useful for the investigation of kinase-engaged signaling pathways and testing of off-target effects of kinase inhibitors.

The Cellular Phosphorylation Assay is a kinase activity assay performed in intact, living cells. The assays have been designed to address compound activity in a physiological environment measuring changes in the phosphorylation status of physiological substrates.

The NanoBRET Intracellular Kinase Assay measures the binding of a compound to the target kinase in intact, living cells. Allowing assessment of target occupancy, binding affinity, and kinetics this assay allows insight into the compound-target interaction in the physiologic environment of cells.