| Assay Method Information | |
| | p-Cl-ANS Fluorescent Binding Assay |
| Description: | Compounds in DMSO (1% final) were added to 384-well black clear bottom plates (Greiner 781091) in 8-point dose response in duplicate using the Echo 550 (Beckman). SU9516 and DMSO were used as controls representing 100% and 0% inhibition of p-Cl-ANS binding, respectively. 20 μL of assay buffer (150 mM NaCl, 50 mM HEPES, pH 7.5 containing 0.01% Triton and 2 mM DTT) was added to each well, the plate was shaken for 1 minute, centrifuged at 2000 rpm for 5 min intervals until air bubbles were eliminated and then read on a CLARIOstar plate reader (BMG; Ex 388 nm, Em 455 nm) to obtain background fluorescence measurements for correction of intrinsic compound fluorescence. Absorbance was then measured at both the excitation and emission wavelengths for correction of the inner filter effect. Next, 5 μL p-Cl-ANS (Wang, N., et al., ACS Omega 2019, 4 (19), 18472-7; 15 μM final) was added to each well in assay buffer. Where present, staurosporine (Enzo Life Sciences; final 5 μM) was added as a premixed solution with p-Cl-ANS. Lastly, 5 uL of 3 μM CDK2 (final 0.5 μM) in buffer was added (final volume of 30 μL) and the plate was shaken, centrifuged, and read as described above. |
| Affinity data for this assay | |
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