| Assay Method Information | |
| | Bcl-2-G101V TR-FRET Assay |
| Description: | Compounds disclosed herein were tested for blocking of Bcl-2-G101V protein with its ligand in an assay based on time-resolved fluorescence resonance energy transfer methodology. 0.1 nM recombinant human Bcl-2-G101V protein was pre-incubated with a serial dilution of compounds disclosed herein (top final concentration is 10 uM or 1 uM or 0.1 uM, 4-fold or 3-fold serially diluted, 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 5 nM FITC labeled Bak peptide Ac-GQVGRQLAIIGDK(FITC)INR-amide and Mab Anti-6His Tb cryptate Gold was added to plate and further incubated at room temperature for 1 hour. The TR-FRET signals (ex337 nm, em490 nm/520 nm) were read on BMG PHERAstar FSX instrument. The inhibition percentage of Bcl-2-G101V interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 490 nm to that at 520 nm. |
| Affinity data for this assay | |
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