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Assay Method Information |
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| | Homogeneous Time-Resolved Fluorescence Assay |
| Description: | For the assay, 50 mL of a 100-fold concentrated solution of the test compound in DMSO is pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). 2 uL of a solution of c-Met in assay buffer [25 mM Hepes/NaOH, pH 7.5; 5 mM MgCl2; 5 mM MnCl2; 2 mM dithiothreitol; 0.1% (v/v) Tween 20 (Sigma); 0.1% (w/v) bovine serum albumin] are added, and the mixture is incubated for 15 min at 22 C. to allow pre-binding of the test compound to the enzyme before the start of the kinase reaction. Then, the kinase reaction is started by the addition of 3 uL of a solution of adenosine triphosphate (ATP, 16.7 uM; final concentration in the 5 uL assay volume is 10 uM) and substrate (2.27 ug/mL, final concentration in the 5 uL assay volume is 1.36 ug/mL 30 nM) in assay buffer, and the resulting mixture is incubated for a reaction time of 30 min at 22 C. |
| Affinity data for this assay | |
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