| Assay Method Information | |
| | In Vitro Assay Dipeptidyl Peptidase IV. Fibroblast Activation Protein and Prolyl Oligopeptidase |
| Description: | The purpose of this assay is to determine the IC50 of various inhibitors against recombinant human dipeptidyl peptidase IV (DPPIV), fibroblast activation protein (FAP) or prolyl oligopeptidase (PREP). The assays are conducted in the following steps:1 Dissolve the compound in DMSO to a final concentration of 100 mM. From this, prepare a 1 mM stock at pH 7.5 in 50 mM Tris, 140 mM NaCl Buffer (FAP)/pH 7.5 in 25 mM Tris, 250 mM NaCl Buffer/pH 8.0 140 mM NaCl Buffer (PREP).</li><li id="ul0012-0002" num="0365">2. Serial dilute (1:10) the 1 mM compound stocks prepared previously into the appropriate assay buffer (FAP: 50 mM Tris, 140 mM NaCl, pH 7.5/PREP: 25 mM Tris, 0.25 M NaCl, pH 7.5/DPPIV: 25 mM Tris, pH 8.0) to one row of a 96-well plate. 3. Prepare 20× substrate solution (FAP and PREP: 2.5 mM Z-Gly-Pro-AMC (VWR, Cat. No. I-1145.0050BA) in DMSO/DPPIV: 100 mM Gly-Pro-AMC (VWR, Cat. No. 100042-646) in DMSO) by diluting the DMSO stocks into the appropriate assay buffer. 4. Dilute the enzymes into their appropriate assay buffers. The final enzyme concentrations should be 0.1, 1.2, and 0.6 nM for DPPIV, FAP and PREP respectively. Add 180 μL to each well needed in columns 2-10. Column 1 (A,B,C) should be prepared with 200 ul of appropriate assay buffer as control. Column 1 (D,E,F,G,H) should be prepared with 20 ul of appropriate assay buffer and 180 ul enzyme as no inhibitor control. 5. Add 20 μL of the compound of interest from the dilution plate prepared in step 2 to columns 2-10 of the assay plate where appropriate. Each sample should be tested in triplicate. Allow this to incubate for 10 minutes at room temperature, shaking the plate for the first two minutes. 6. Add 10 μL of 20× substrate prepared in step 3 to each well and allow this to incubate for 15 minutes at room temperature, shaking the plate for the first two minutes. 7. Read the fluorescence at λex: 380, λem: 460. |
| Affinity data for this assay | |
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