| Assay Method Information | |
| | BCL2 WT, BCL2 G101V, BCL2 D103Y, BCL2 F104I, BCL2 D111A and BCLXL WT TR-FRET Binding AssayBCL2 WT, BCL2 G101V, BCL2 D103Y, BCL2 F104I, BCL2 D111A and BCLXL WT TR-FRET Binding Assay |
| Description: | The assay was performed in a 40 μL volume in white 384-standard well plates (Corning #3574). An 11-point serial dilution of each compound was prepared in DMSO and transferred directly to plate followed by addition of fluorescein labelled peptide and lastly protein. Final assay conditions were 0.5 nM BCL2_wt, BCL2_G101V, BCL2_D103Y, BCL2_F104I, BCL2_D111A or BCLXL_wt, 2 or 20 nM fluorescein labelled peptide, 50 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM TCEP (Tris(2-carboxyethyl)phosphine), 0.05% Tween 20, 5% DMSO. The mixture was incubated for 2 hours at 23° C. TR-FRET measurements were performed on a Biotek Synergy Neo plate reader. TR-FRET was measured by excitation of the Terbium-donor at 340 nm and subsequent (delay time 100 μs) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a collection time of 300 μs. The TR-FRET signal was calculated as the emission-ratio at 520 nm over 495 nm. |
| Affinity data for this assay | |
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