| Assay Method Information | |
| | Inhibition Assay |
| Description: | Prepare a working solution of 100 μM Amplex® Red reagent containing 0.25 U/mL HRP, 0.08 U/mL L-glutamate oxidase, 0.5 U/mL 1-glutamate-pyruvate transaminase, 200 μM L-alanine and keep protected from light. Add 10 μl/well of the PSMA solution at 25 nM. Final concentration is 5 nM. Transfer 10 μl/well of the inhibitor solution previously prepared in a separate 96 wells/plate. Incubate PSMA and inhibitors for 20′ at 37° C. in the dark After, add 10 μl of substrate solution NAAG to have a final concentration of 40 μM.Pipette 20 μl/well of the working solution prepared above. Incubate the reactions for 30 min at 37° C., protected from light. Because the assay is continuous (not terminated), fluorescence may be measured at multiple time points to follow the kinetics of the reactions. Measure the fluorescence in a fluorescence microplate reader using excitation in the range of 530-560 nm and emission detection at ˜590 nm. Ec50s were determined with GraphPad Prism. Ki is calculated based on Cheng-Prusoff equation. |
| Affinity data for this assay | |
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