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Assay Method Information

Assay Name:  TR-FRET Competition Assay
Description:  The experiments were performed in 384-well black small volume microtiter plates(Greiner) in a buffer composed of 50mM Hepes, pH 7.5 (Applichem), 50 mM NaCl (Sigma), 400 mM KF (Sigma), 0.5 mM CHAPS (Sigma), and 0.05% BSA (Sigma) in a final volume of 5-10 μl (n = 4). BRD4 (100 nM) protein domains were mixed with 200 nM concentrations of the biotin-labeled histone peptides and incubated for 30 min at room temperature. The protein-peptide complexes at equilibrium were then detected with 5 nM Eu3+ cryptate-conjugated streptavidin (CisBio) and 10 nM anti-His6-XL665 (Cisbio). To analyze the binding of mutated histone peptides at different ionic strengths, streptavidin Eu33+ chelate (W1024, PerkinElmer Life Sciences) was used instead of cryptate as fluorescence donor, and KF was replaced by NaCl at concentrations of 20, 100, and 500 mM. After 3 h of further incubation at 4 °C, the plates were measured in a PheraStar reader (BMG Labtech) using the homogeneous time-resolved fluorescence module (excitation, 337 nm with 10 flashes; emission, 620 and 665 nm). For competition assays 50 nM BRD4 BD1 and 500 nM BD2 protein were preincubated with serial dilutions of the unlabeled H4 (1-25) K5(ac)K8(ac)K12(ac)K16(ac) tetra-acetylated peptide(15 nM to 250 μM, 2-fold) or with JQ1 (0.61 nM to 10 μM, 2-fold) for 30 min at room temperature. The plates were then processed as described above.
Affinity data for this assay
 

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Last update November 1, 2007
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