| Solution Information | help | |
| Enzyme: | Procathepsin L | |
| inhibitor: | BDBM41147 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials Human liver cathepsin L was purchased from Calbiochem (Cat #219402). Substrate Z-Phe-Arg-AMC was from Sigma-Aldrich. Assay buffer consisted of 20 mM sodium acetate, pH 5.5, containing 1 mM EDTA, and 5 mM DTT. Low-volume 384-well black plates were from Corning (Item #3676). Assay Cathepsin L (8.7 ng/mL) was incubated with Z-Phe-Arg-AMC substrate (1 uM) in 10 uL of assay buffer (see above) for 1 hr at room temperature. HTS actives were confirmed by IC50 determination as described below. IC50 protocol 1. Serial dilute compounds at 50x concentration in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM) 2. Fill Corning low-volume 384-well black plate with 4 uL water using Multidrop-micro 3. Add 5 uL assay buffer to columns 1 and 23 using Multidrop-384 4. Add 200 nL of compound (in DMSO from step 1) using Evolution pintool 5. Add 1 uL of Z-Phe-Arg-AMC substrate (10 uM in 5x assay buffer) using Multidrop-micro 6. Add 5 uL enzyme (17.4 ng/mL in assay buffer) using Multidrop-384 | |
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