NanoBRET™ Target Engagement HDAC Assays
Directly Measure HDAC-Test Compound Affinity and Residence Time in Live Cells
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NanoBRET™ Technology for Target Engagement
The NanoBRET™ Target Engagement (TE) Assay measures compound binding at select target proteins in intact cells, in real time. 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 tracer that specifically binds to the target protein; a substrate for NanoLuc® luciferase; and a cell-impermeable inhibitor for NanoLuc® luciferase.
The assay uses bioluminescence resonance energy transfer (BRET), achieved by transferring the luminescent energy from NanoLuc® luciferase to the fluorescent tracer that is bound to the target protein-NanoLuc® fusion. This energy transfer makes it possible to directly measure compound binding affinity as well as compound-target residence time.
Compounds that are applied to the cells and specifically engage the intracellular target protein-NanoLuc® fusion will result in a decrease in BRET. To ensure accurate assessment of intracellular target engagement, a NanoLuc® inhibitor is used to mitigate any extracellular NanoLuc® signal that may arise from cells compromised during handling, while not adversely affecting NanoLuc® luciferase expressed within healthy living cells
