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.
Surelight allophycocyanin streptavidin
Manufactured by PerkinElmer
SureLight allophycocyanin-streptavidin is a fluorescent label used in immunoassay and flow cytometry applications. It consists of the naturally fluorescent protein allophycocyanin conjugated to the biotin-binding protein streptavidin. This label can be used to detect and quantify biotinylated targets.
Automatically generated - may contain errors
Lab products found in correlation
Lanthascreen eu anti his tag antibody, by Thermo Fisher Scientific (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)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 protocols using surelight allophycocyanin streptavidin
1
HTRF Assay for YEATS and BRD4 Binding
2
Binders Screening for Epigenetic Targets
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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.
3
Syk Kinase Phosphorylation Assay
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Example 428
Phosphorylation of a peptide substrate by purified SYK kinase was measured using a FRET method. Following dilution in 30% DMSO, 5 μl compound was added to 45 ul reaction mix such that the final concentrations were 50 mM Tris pH 7.5, 5 mM MgCl2, 4.7 nM syk enzyme (ProQuinase), 100 μM DDT, 100 μM ATP, and 545 nM biotin-DEEDYESP-OH substrate. Plates were incubated at room temperature for 1 hour and 50 μl detection buffer (100 mM Tris, pH 7.5, 300 mM NaCl2, 20 mM EDTA, 0.02% Brij 35, 0.05% BSA (Sigma), 46 nM SureLight Allophycocyanin-Streptavidin (Perkin Elmer), and 21 nM LANCE Eu-W1024 Anti-phosphotyrosine (Perkin Elmer)) added. Plates were incubated for a further 1 hour at room temperature and substrate phosphorylation determined using an Analyst (Molecular Devices) with the settings at Ex 360, Em 665, 60 μsec delay.
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