| Assay Method Information | |
| | c-MET Inhibitory Activity |
| Description: | Experimental methods: Enzyme reaction substrate, poly(Glu, Tyr)sodium salt (Glu:Tyr=4:1) was diluted with potassium-free phosphate buffered saline (PBS) (10 mM sodium phosphate buffer, 150 mM NaCl, pH=7.2-7.4) to 20 μg/mL, coated on an ELISA plate with a volume of 125 μL/well, and reacted at 37° C. for 12 h. The supernatant in each well was discarded, and the ELISA plate was washed with 200 μL/well T-PBS (potassium-free PBS containing 0.1% Tween-20) three times for 5 minutes each time. Then the ELISA plate was dried in an oven at 37° C. for 2 h.The maximum concentration of compounds was set to 3.0 μM, and the compound solution was subjected to 3-fold dilution with DMSO, a total of 10 concentration levels were obtained as follows: 3.0 μM, 1.0 μM, 0.33 μM, 0.11 μM, 0.037 μM, 0.012 μM, 0.0041 μM, 0.0014 μM, 0.00045 μM and 0.00015 μM. Each concentration was tested in triplicate. 80 μL of Adenosine Triphosphate (ATP) solution diluted with reaction buffer, 10 μL of compounds with various concentrations (10 μL blank DMSO solution was added to a negative control well) and 10 μL of enzyme solution diluted with reaction buffer were sequentially added into each well. The mixture was then processed on a shaker at 37° C. for 1 h. The final reaction buffer contained HEPES (pH=7.4) 50 mM, MgCl 2 50 mM, MnCl2 0.5 mM, Na3 VO4 0.2 mM and DTT 1 mM. The final concentration of the ATP solution was 4 μM, and the amount of enzyme was 1 μL/well. The ELISA plate was washed three times with T-PBS. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |