A N-terminal NanoLuc® luciferase ID1 fusion protein was synthesized by Genewiz (South Plainfield, NJ, USA) into the pUC57 backbone and then cloned into the pcDNA3.1 plasmid with EcoRI-HF and XbaI (both enzymes from New England Biolabs, Ipswich, MA, USA). The assay was performed essentially as described in the NanoBRET™ TE Intracellular BET BRD Assay kit manual (Promega Corporation, Madison, WI, USA). Briefly cells were transfected and after 24 hours were plated onto flat bottom, non-binding surface, white, polystyrene 96-well plates (Corning Incorporated, Kennebunk, ME, USA). The AGX51 tracer was then added (0–4 μM), followed by digitonin (Sigma, St. Louis, MO, USA) to permeabilize the cells (50 μg/mL). The NanoBRET™ Nano-Glo® Substrate (Promega) was then added and readings taken using a GloMax Discover System instrument (Promega). For the competition assays, cells were treated with 2 μM of AGX51 tracer and 0–60 μM of AGXA or AGX51.
Glomax discover system instrument
Manufactured by Promega
The GloMax Discover System is a multi-mode microplate reader instrument that measures luminescence, fluorescence, and absorbance in microplates. The instrument provides high-sensitivity detection for a variety of assays, including cell-based, enzymatic, and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Digitonin, by Merck Group (2 mentions)
Nanobret nano glo substrate, by Promega (2 mentions)
N terminal nanoluc luciferase id1 fusion protein, by Genewiz (2 mentions)
Nanobret te intracellular bet brd assay kit, by Promega (2 mentions)
Ecori hf, by New England Biolabs (2 mentions)
Xbai, by New England Biolabs (2 mentions)
2 protocols using glomax discover system instrument
1
NanoBRET Intracellular BET BRD Assay
2
NanoBRET Intracellular BET BRD Assay
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A N-terminal NanoLuc® luciferase ID1 fusion protein was synthesized by Genewiz (South Plainfield, NJ, USA) into the pUC57 backbone and then cloned into the pcDNA3.1 plasmid with EcoRI-HF and XbaI (both enzymes from New England Biolabs, Ipswich, MA, USA). The assay was performed essentially as described in the NanoBRET™ TE Intracellular BET BRD Assay kit manual (Promega Corporation, Madison, WI, USA). Briefly cells were transfected and after 24 hours were plated onto flat bottom, non-binding surface, white, polystyrene 96-well plates (Corning Incorporated, Kennebunk, ME, USA). The AGX51 tracer was then added (0–4 μM), followed by digitonin (Sigma, St. Louis, MO, USA) to permeabilize the cells (50 μg/mL). The NanoBRET™ Nano-Glo® Substrate (Promega) was then added and readings taken using a GloMax Discover System instrument (Promega). For the competition assays, cells were treated with 2 μM of AGX51 tracer and 0–60 μM of AGXA or AGX51.
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