| Assay Method Information | |
| | Affinity Assays Using Enzyme Activity Assay |
| Description: | Buffer 1 (50 mM HEPES, 0.1 M NaCl, pH 7.5) was used to prepare a 0.4 μg/mL rhPSMA solution and a 40 μM solution of the substrate N-Acetyl-Asp-Glu. rhPSMA was mixed with the small molecules to be tested in a 96-well plate, with a constant rhPSMA content of 50 ng/well maintained. Meanwhile, the small molecules were step-wise diluted to final concentrations of 1 μM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.41 nM, 0.137 nM, 0.045 nM, and 0 nM. In addition, a positive control was set up using PSMA-617. The rhPSMA-small molecules were taken at 40 μL/well and well mixed with the 40 μM solution of the substrate N-Acetyl-Asp-Glu (40 μL/well). The mixtures were incubated at 37° C. in the dark for 1 h, were heated at 70° C. for 5 min to quench the reactions, and were cooled to room temperature. Buffer 2 (0.2 M NaOH, 0.1% beta-Mercaptoethanol) was used to prepare a 15 mM OPA solution. The OPA solution was added to the reaction systems at 80 μL/well and well mixed, and then the plate was incubated at room temperature for 10 min. The mixtures were taken at 100 μL/well and added to a 96-well Flat Black. With the excitation wavelength set to 330 nm and the emission wavelength to 465 nm, the intensity of signals was measured. |
| Affinity data for this assay | |
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