All HTRF assays were performed by combining recombinant protein and synthetic histone peptide in assay buffer (25 mM HEPES pH 7, 20 mM NaCl, 0.2% Pluronic F-127, and 0.05% BSA) with 1 nM LanthaScreen Eu-anti-His Tag antibody (ThermoFisher, Cat. No, PV5597) and 8.9 nM SureLight allophycocyanin-streptavidin (Perkin Elmer, APC-SA, Cat. No. CR130-100). ENL YEATS, AF9 YEATS, and YEATS4 were used at 5 nM and BRD4 BD1 was used at 10 nM. ENL and AF9 assays were performed with H3(13-32)K27cr (13.3 and 100 nM, respectively), custom synthesized at ABclonal (N-terminal biotin, C-terminal amide); YEATS4 assay was performed with 25 nM H3(21-43)K27ac from Anaspec (Cat. No. AS-64846-1); and BRD4 BD1 assay was performed with 13.3 nM tetra-acetylated H4 (BioVision, Cat. No. 7144-01). Once all reagents were combined (with or without peptide), 10 μL was dispensed per well into black 384-well low-volume plates (Corning, Cat. No. 3821) and drug was added by pin tool transfer (Biomek FX). Assays were incubated for 2 or more hours before measurement of HTRF signal on a PHERAstar plate reader (BMG Labtech; simultaneous dual emission; excitation = 337 nm, emission 1 = 665 nm, emission 2 = 620 nm). HTRF signal (ratio of emission 1 to emission 2) from vehicle-treated wells (maximum signal) and no-peptide-control wells (minimum signal) were used to calculate percent inhibition for drug-treated wells.
Lanthascreen eu anti his tag antibody
Manufactured by Thermo Fisher Scientific
The LanthaScreen Eu-anti-His Tag antibody is a fluorescent-labeled antibody that specifically binds to the His-tag epitope. It is designed for use in fluorescence-based detection and characterization assays, such as protein-protein interaction studies and high-throughput screening applications.
Automatically generated - may contain errors
Lab products found in correlation
Surelight allophycocyanin streptavidin, by PerkinElmer (2 mentions)
Tetra acetylated h4, by Abcam (2 mentions)
Pherastar plate reader, by BMG Labtech (2 mentions)
Black 384 well low volume plates, by Corning (1 mentions)
Echo liquid handler, by Beckman Coulter (1 mentions)
Hibase, by Greiner (1 mentions)
2 protocols using lanthascreen eu anti his tag antibody
1
HTRF Assay for YEATS and BRD4 Binding
2
Binders Screening for Epigenetic Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
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AF9 YEATS, BRD4 BD1, and ENL YEATS HTRF assays
were performed exactly as previously described.33 (link) All assays were conducted by combining recombinant AF9
YEATS (5 nM), BRD4 BD1 (10 nM), or ENL YEATS (5 nM) with synthetic
histone peptide (100 nM H313–32K27cr custom synthesized
by ABclonal, 13.3 nM tetra-acetylated H4 from BioVision Cat. No. 7144–01,
or 13.1 nM H313–32K27cr, respectively), 1 nM LanthaScreen
Eu-anti-His Tag antibody (ThermoFisher, PV5597), and 8.9 nM SureLight
allophycocyanin-streptavidin (PerkinElmer, CR130–100). For
the primary screen, the assay mixture was dispensed into black 1536-well
plates (Greiner HiBase) at 6 μL/well and compounds were added
by acoustic transfer (Labcyte, Echo Liquid Handler). Dose–response
assays were performed with 10 or 20 μL assay volumes in black
384-well low-volume plates (Corning, Cat. No. 3821), and compounds
were added by pin tool transfer (Biomek FX). Compounds were incubated
with the assay mixture for at least 2 h before measuring the HTRF
signal on a PHERAstar plate reader (BMG Labtech; simultaneous dual
emission; excitation = 337 nm; emission 1 = 665 nm; emission 2 = 620
nm). The HTRF signal was calculated as the ratio of emission 1 to
emission 2. Each plate included samples treated with vehicle (DMSO)
or lacking the peptide substrate, which were used to calculate the
percent inhibition for compound treated wells.
were performed exactly as previously described.33 (link) All assays were conducted by combining recombinant AF9
YEATS (5 nM), BRD4 BD1 (10 nM), or ENL YEATS (5 nM) with synthetic
histone peptide (100 nM H313–32K27cr custom synthesized
by ABclonal, 13.3 nM tetra-acetylated H4 from BioVision Cat. No. 7144–01,
or 13.1 nM H313–32K27cr, respectively), 1 nM LanthaScreen
Eu-anti-His Tag antibody (ThermoFisher, PV5597), and 8.9 nM SureLight
allophycocyanin-streptavidin (PerkinElmer, CR130–100). For
the primary screen, the assay mixture was dispensed into black 1536-well
plates (Greiner HiBase) at 6 μL/well and compounds were added
by acoustic transfer (Labcyte, Echo Liquid Handler). Dose–response
assays were performed with 10 or 20 μL assay volumes in black
384-well low-volume plates (Corning, Cat. No. 3821), and compounds
were added by pin tool transfer (Biomek FX). Compounds were incubated
with the assay mixture for at least 2 h before measuring the HTRF
signal on a PHERAstar plate reader (BMG Labtech; simultaneous dual
emission; excitation = 337 nm; emission 1 = 665 nm; emission 2 = 620
nm). The HTRF signal was calculated as the ratio of emission 1 to
emission 2. Each plate included samples treated with vehicle (DMSO)
or lacking the peptide substrate, which were used to calculate the
percent inhibition for compound treated wells.
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