RECENT CITATIONS 9H-purine scaffold reveals induced-fit pocket plasticity of the BRD9 bromodomain. S. Picaud et al. (2015) J. Med. Chem. 58(6): 2718-36. T he authors used bioluminescence resonance energy transfer (BRET) to test the ability of a bromodomain 9 ligand to disrupt binding to histone. HEK293 cells were cotransfected with a histone H3.3-HaloTag® fusion vector and either NanoLuc® NanoLuc® -BRD9 bromodomain or -full-length BRD4 fusion vector. After 24 hours, the transfected cells were trypsinized, diluted in phenol red-free DMEM with or without 10nM of HaloTag® NanoBRET™ Nano-Glo® Substrate (final concentration 10μM) was added. Fluorescence was measured and a corrected BRET ratio calculated. Cytotoxicity was assessed after the NanoBRET™ cells with the CellTiter-Glo® assay by incubating the Reagent for 30 minutes and measuring luminescence. To examine histone H3.3 localization, HEK 293 cells were transfected with the histone H3.3HaloTag® fusion vector using FuGENE® 618 Ligand and dispensed into a 96-well plate. One of two potential BRDdisrupting compounds, 7d or 11, was added to a final concentration of 0.005–33μM, cells were incubated for 18 hours and NanoBRET™ HD Transfection Reagent. After 24 hours, cells were labeled with 5μM HaloTag® TMR ligand for 15 minutes, washed with complete medium, incubated for 30 minutes and imaged with a confocal microscope. More » © 2015 Promega Corporation. CellTiter-Glo, FuGENE, HaloTag, Nano-Glo, NanoLuc and QuantiFluor are registered trademarks of Promega Corporation. NanoBRET, Quantus and RealTime-Glo are trademarks of Promega Corporation. Products may be covered by pending or issued patents or may have certain limitations. Please visit www.promega.com for more information. Contact us enews | May 2015 Pagina 17
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