| Assay Method Information | |
| | FAP Protease Activity Assay |
| Description: | Recombinant mouse FAP (R&D systems, #8647-SE) was diluted in assay buffer (50 mM Tris, 1 M NaCl, 1 mg/mL BSA, pH 7.5) to a concentration of 3.6 nM. 25 ul of the FAP solution was mixed with 25 ul of a 3-fold serial dilution of the test compounds and incubated for 5 min in a white 96-well ProxiPlate (Perkin Elmer). As specific FAP substrate the FRET-peptide HiLyteFluor 488-VS (D-) P SQG K (QXL 520)NH2 was used (Bainbridge, et al., Sci Rep, 2017, 7:12524). 25 uL of a 30 uM substrate solution, diluted in assay buffer, was added. All solutions were equilibrated at 37 C. prior to use. Substrate cleavage and increase in fluorescence (excitation at 485 nm and emission at 538 nm) was measured in a kinetic mode for 5 minutes at 37 C. in a SPECTRAmax M5 plate reader. RFU/sec was calculated by SoftMax Pro software and plotted against test compound concentration. |
| Affinity data for this assay | |
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