| Assay Method Information | |
| | Fluorescence Polarization Assay |
| Description: | Bcl-xL and Bcl-2 FP binding affinity of compound(s) of Formulae (I), (I-a), (I-b), (I-c), (I-d) and/or (I-c) may be determined using a variety of known methods. One such assay is a sensitive and quantitative in vitro binding assay using fluorescence polarization ("FP") described by Wang, J.-L.; Zhang, Z-J.; Choksi, S.; Sjam. S.; Lu, Z.; Croce, C. M.; Alnemri, E. S.; Komgold, R.; Huang, Z. Cell permeable Bcl-2 binding peptides: a chemical approach to apoptosis induction in tumor cells. Cancer Res. 2000, 60, 1498-1502). Additionally, the binding affinity of compound(s) of Formulae (I), (I-a), (I-b), (I-c), (I-d) and/or (I-e) to Bcl-2 protein in vitro was determined by a competitive binding assay based on fluorescence polarization. For example, fluorescence polarization ("FP")assays may be developed using a c-terminal 6XHIS tagged Bcl-2 (aa 1-204) and a C-terminal 6XHIS tagged Bcl-XL (aa 1-209). The tracer may be a synthetic peptide BH-3 peptide Bim conjugated to Fluorescein isothiocyanate (FITC-DLRPEIRIAQELRRIGDEFNETYTRR). Dilutions of either Bcl-2 (1.3 nM) or Bcl-XL (0.8 nM) may be added to serial dilutions of antagonist and incubated for one hour prior to the addition of 2 nM of fluorescent peptide tracer (Anaspec, Fremont, Calif.) in the assay buffer. Final assay buffer conditions may be 20 mM HEPES, pH 7.5, 1 mM DTT, 0.005% Tween-20 and 50 mM NaCl. Samples may be read after 20-minute incubation. Fluorescence polarization values may be plotted as a function of the antagonist concentration. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |