Cell-based assays are useful for secondary kinase screening & profiling after initial compound discovery as they test occupancy, selectivity, and affinity within the cellular environment, where engagement naturally occurs. Some small molecule compounds have been shown to be effective in a biochemical assay but fail in subsequent testing in cell-based assays. Even more surprising are compounds that would have been discarded after failure in biochemical assays, but they showed to work in the cell.
Cell-based screening assays provide a more accurate environment like local ion concentrations, compartments with ideal pH, cofactor availability, membrane barriers, and potential cytotoxic effects or off-target effects – all essential to successful drug discovery. Our portfolio covers the NanoBRET™ TE assay, a tyrosine kinase assay, and cell lines for protein-protein interaction detection.
NanoBRET™ TE Intracellular Kinase Assay (HEK293 cells) I Kinase Screening & Profiling
This assay uses bioluminescence resonance energy transfer (BRET) to monitor the activity of kinases in real-time. In the assay, a substrate for the kinase of interest is labeled with two different fluorescent proteins, a NanoLuc® luciferase and a fluorescent protein (NanoBRET™ tracer). When the kinase phosphorylates the substrate, the fluorescent proteins are brought into proximity, which results in energy transfer and a BRET signal. Binding the test compound to the target protein results in a loss of BRET signal between the target protein and the tracer. As a result, the NanoBRET™ TE assay measures the apparent affinity of test compounds by competitive displacement of the NanoBRET™ tracer, reversibly bound to the NanoLuc® luciferase-kinase fusion protein.
This assay allows for real-time monitoring of kinase activity inside living cells, making it a valuable tool for kinase profiling & screening, studying compound affinity and fractional occupancy for kinases under more physiologically relevant information. This assay detects multiple kinase inhibitor types, such as inhibitors with different binding modes (including type I, II, allosteric and covalent). In addition to equilibrium evaluation, it is also possible to obtain the durability of the test compound binding to a kinase (Residence Time), enabling better compound optimization.
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Tyrosine Kinase Cell-Based Assays (BA/F3 cells) I Kinase Screening & Profiling
In cooperation with Advanced Cellular Dynamics (ACD, Seattle, WA, USA), we offer a unique cell-based tyrosine kinase assay panel. This service provides tyrosine kinase screening & profiling using transfectants in which activated recombinant kinases are expressed and may help you to discover direct inhibitors to target tyrosine kinases.
The assay relies on constitutively activated kinases providing cytokine-independent growth to IL3-independent Ba/F3 (Pro-B) cell line. The cells are transformed by inducing target genes via viral vectors. The activity of the transformed kinase overrides IL-3 dependency for cellular proliferation and survival. If the kinase inhibitor (compound) specifically blocks the activity of the recombinant kinase, the modified cells undergo programmed cell death (apoptosis), resulting in a lower measurement of residual ATP.
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Protein-Protein Interaction (PPI) Detection Assay
Protein-protein interaction detection is the process of identifying and characterizing physical associations between two or more proteins in a biological system. Our split luciferase complementation assay utilizing a unique luciferase derived from Pyrearinus termitilluminans (Emerald Luciferase, E-Luc) is a valuable tool for your study of Protein-Protein Interactions (PPI). This enables the detection of various types of PPIs, including GPCRs, with ease and high sensitivity. In addition to established cell lines on the list, we develop custom stable transfected cell lines suitable to detect specific PPIs of your interest! We also develop transfectant cell lines suitable for custom assay on a fee-for-service basis. The cell assay reagent (Split Glow) will be included with each purchase of a stable cell line.
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