| Assay Method Information | |
| | Enzyme Linked Immunosorbant Assay |
| Description: | (1) The enzyme reaction substrate Poly(Glu, Tyr) 4:1 was diluted to 20 ug/mL, 1254/well coated enzyme label plate with potassium ion-free PBS (10 mM sodium phosphate buffer, 150 mM NaCl, pH7.2-7.4), which was placed at 37 C. to allow to react for 12-16 h. The liquid in the wells was discarded. The plate was washed with 200 uL/well of T-PBS (potassium ion-free PBS containing 0.1% Tween-20) for three times (each for 5 min). The enzyme label plate was placed into a drying oven at 37 C. to dry for 1-2 h.(2) To each well was added 504 of ATP solution diluted with reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT), and to each well was added 1 uL of the compounds. When adding the test compounds, 6 concentration gradients were set for each compound (the starting concentrations and dilution times were determined according to the results of preliminary screening so as to ensure that the maximum inhibition ratio was about 80%. |
| Affinity data for this assay | |
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