| Assay Method Information | |
| | Inhibition Assay |
| Description: | Inhibition assay of FAP. The compounds were precisely weighed (range between 20-1000 pg) on a micro-scale (HuberLab, Sartorius QUINTIX35-1S) and dissolved in DMSO to prepare stock solutions at 1 mM. Stock solutions were further diluted with FAP activation buffer (50 mM Tris, 100 mM NaCl, 1 mM EDTA, pH=7.4) to a final DMSO concentration of 10% v/v. 10 μL of each inhibitor were serially diluted in a 384 well plate (grenier-bio one, PS, F-bottom, Black, non-binding) with 10 μL of 10% DMSO FAP buffer (1:1 dilution). FAP (human and murine) was diluted to the desired concentration with FAP activation buffer and added to the serial dilution of inhibitor (10 μL for each well). Each protein-inhibitor solution was incubated for 30 minutes at room temperature. Each measurement was performed with an enzyme concentration (FAP) lower than the expected IC50. A 60 μM solution of substrate (Z-Gly-Pro-AMC) in FAP activation buffer (<1% DMSO) was added to the plate (10 μL for each well). The reaction was incubated from 1 minute to 48 hours at room temperature (22° C.-25° C.). The emission was read at 465 nm (excitation wavelength 360 nm) for 40 μs by Tecan Spark (s.n. 1808004082). The following controls were added to each row: positive control (no inhibitor control, 10 μL of 10% DMSO FAP buffer+10 μL of FAP solution+10 μL of substrate solution), negative control (no protein control, 10 μL of 10% DMSO FAP buffer+10 μL of FAP activation buffer+10 μL of substrate solution). |
| Affinity data for this assay | |
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