| Assay Method Information | |
| | FRET Assay |
| Description: | Compounds of the invention were dissolved in DMSO at a concentration of 3 mM with subsequent dilutions in assay buffer (50 mM HEPES PH7.0, 150 mM NaCl, 0.05% BSA, 0.2% Pluronic F-127) such that the assay contained 1% DMSO. In a white 384 shallow well Microplate (Proxiplate-384 Plus, PerkinElmer, 6008280), 150 nL of compound or vehicle (1% DMSO in assay buffer) for the high control (HC) wells and 5 μL of 30 nM ENL Protein (6×HIS ENL YEATS Domain, EpiCypher, 15-0069) were combined and incubated 15 minutes at RT. Low control (LC) wells received 5 μL of assay buffer instead of ENL protein. Then 5 μL of 15 nM H3K9cr peptide (H3 aa1-20, biotinylated; EpiCypher, 12-0099) in assay buffer was added and incubated 30 minutes at RT. Finally a 5 μL mix of 45 nM Anti-6HIS ULight (PerkinElmer, TRF0105) and 1.5 nM Streptavidin-Europium Chelate (PerkinElmer, AD0060) were added and incubated for a further 30 minutes at RT. The TR-FRET signal (665 nm signal/615 nm signal X 10,000) was measured using a PerkinElmer 2104 EnVision (Xenon Flash Lamp excitation, 320 nm±37.5 nm excitation filter, 407 nm cut off dichroic mirror, 615 nm±4.25 (Europium) nm and 665 nm±3.75 nM (ULight) emission filters). Compound concentration response curves were performed in duplicate over the concentration range of 0.15 nM-30 μM. The response at each compound concentration minus the LC value was converted to percent inhibition of the vehicle control group response (HC-LC). The relationship between the % inhibition and the compound concentration was analyzed using a four parameter logistic equation to estimate lower and upper asymptotes, the compound concentration producing 50% inhibition (IC50 value) and the slope at the mid-point location. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |