| Assay Method Information | |
| | rhFAP Inhibition Assay Protocol |
| Description: | 1) DMSO stocks (1 or 10 mM) were diluted 1:100 into assay buffer (Dulbecco's PBS without calcium or magnesium; Gibco, Cat. No. 14190136) to yield 10 or 100 mM working stocks for the dilution plate.2) The dilution plate was prepared by adding the 10 or 100 mM stocks prepared in the previous step to row A of a 96-well plate. From this, 1:10 serial dilutions into assay buffer were performed for rows B—H.3) Recombinant human FAP (rhFAP from R&D Systems, Cat. No. 3715-SE) was diluted into assay buffer for a working enzyme concentration of 0.22 nM (1.1×). All wells in columns 2-10 of each black, clear-bottom assay plate (Falcon, Cat. No. 353219) received 180 mL of FAP. Wells in column 1 served as the controls for the assay.a. Background controls: wells A1-C1 (200 mL assay buffer alone)b. No inhibitor controls: wells D1-F1 (180 mL FAP+20 mL assay buffer alone)4) Enzyme pretreatment with inhibitors was carried out via addition of 20 mL of the inhibitor of interest from the dilution plate prepared in step 2 to columns 2-4, 5-7 or 8-10 of the assay plate. This allowed for testing of all inhibitors in triplicate. Inhibitors were incubated with the enzyme for 10 min at 37° C.5) A 500 mM (20×) 3144-AMC substrate (10 mM DMSO stock, Tufts University Batch ID3144-03) working solution was prepared via dilution of the DMSO stock into assay buffer. All wells were treated with 10 mL of the working solution for a final assay concentration of 25 mM. The plates were shaken briefly and incubated for 30 min at 37° C.6) Endpoint fluorescence was measured at Ex380:Em460. Data was analyzed using GraphPad Prism to calculate IC50 values which are shown |
| Affinity data for this assay | |
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