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NanoBRET TE Intracellular Kinase Assay, K-12

by Promega
€ 0.00
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The NanoBRET Target Engagement (TE) Assays measure compound binding at select target proteins within intact cells. This target engagement assays are based on the NanoBRET System, an energy transfer technique designed to measure molecular proximity in living cells. The NanoBRET TE Intracellular Kinase Assays measure the apparent affinity of test compounds by competitive displacement of the NanoBRET Tracer reversibly bound to a NanoLuc luciferase fusion protein in cells. In the first step of the NanoBRET TE Assay, a fixed concentration of tracer is added to cells expressing the desired NanoLuc fusion protein to generate a BRET reporter complex. Introduction of competing compounds results in a dose-dependent decrease in NanoBRET energy transfer, which allows quantitation of the intracellular affinity of the target protein for the test compound.The NanoBRET TE Assay has been applied successfully to study multiple target classes including histone deacetylases and the BET family of the bromodomains. Here, we describe the NanoBRET TE Intracellular Kinase Assay, which measures compound binding to a kinase protein-NanoLuc luciferase fusion. The largest group of enzymes in the human proteome, the kinases, are essential to myriad cellular processes from regulation of cell physiology to signal transduction. As such, dysregulation of kinase activity is associated with a variety of human diseases, including cancers and autoimmune diseases, and kinases have been established as promising drug targets for therapeutic intervention. The NanoBRET TE Intracellular Kinase Assays, K-4 and K-5, allow quantitation of intracellular affinity for a test compound to a diverse set of full-length protein kinases expressed inside living cells.The NanoBRET TE Assay uses four key components: an expressed cellular target protein that is fused to the bright NanoLuc luciferase; a cell-permeable fluorescent NanoBRET tracer that specifi cally binds to the target protein; a substrate for NanoLuc luciferase; and an extracellular inhibitor for NanoLuc luciferase. Bioluminescence resonance energy transfer (BRET) is achieved through a nonradiative transfer of the luminescent energy from NanoLuc luciferase to the fluorescent tracer that is bound to the target protein-NanoLuc fusion. Compounds that are applied to the cells and specifically engage the intracellular target protein-NanoLuc(R) fusion will result in a decrease in BRET. To ensure accurate assessment of intracellular target engagement, an extracellular NanoLuc inhibitor is used to mitigate any NanoLuc luciferase signal that may arise from cells compromised during handling, while not adversely affecting NanoLuc luciferase expressed within healthy living cells. The NanoBRET TE Assays have been optimized to use a blue-shifted NanoLuc donor and a red-shifted fluorescent tracer acceptor (NanoBRET 590) that have minimal spectral overlap within the assay . This results in an optimized signal:background ratio and hence an optimized NanoBRET ratio.

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