| Assay Method Information | |
| | Bcl-2 TR-FRET Assay |
| Description: | Compounds disclosed herein were tested for blocking of Bcl-2 protein with its ligand in an assay based on Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) methodology. Recombinant human 0.05 nM Bcl-2 protein was pre-incubated with a serial dilution of compounds disclosed herein (top final concentration is 1 uM or 0.1 uM or 0.02 uM or 0.01 uM, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 mM potassium phosphate buffer, pH 7.5, 50 mM NaCl. 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK(FITC)INR-amide (0.5 nM) and MAb Anti 6His Tb cryptate Gold were added to plate and further incubated at room temperature for 1 hour. The TR-FRET signals (337 nm-520 nm-490 nm) were read on BMG PHERAstar FSX instrument. The inhibition percentage of Bcl-2 interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the TR-FRET signals. |
| Affinity data for this assay | |
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