| Solution Information | help | |
| Enzyme: | Cathepsin G | |
| inhibitor: | BDBM41385 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials Human neutrophil cathepsin G was purchased from Calbiochem (Cat #219373). Substrate Suc-Ala-Ala-Pro-Phe-AMC was from Bachem (Cat #I-1465.0050). Assay buffer consisted of 100 mM HEPES, pH 7.4, 0.5 M sodium chloride. Low-volume 384-well black plates were from Corning (Item #3676). Assay Cathepsin G (4.2 ug/mL) was incubated with Suc-Ala-Ala-Pro-Phe-AMC substrate (15 uM) in 10 uL of assay buffer (see above) for 3 hr at room temperature. Activity of single compounds identified from mixture HTS were confirmed by IC50 determination as described below. IC50 protocol 1.Serial dilute single compounds at 50x concentration in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM) 2.Fill low-volume 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 Suc-Ala-Ala-Pro-Phe-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro 6.Add 5 uL enzym | |
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