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| Solution Information | help | |
| Enzyme: | Kallikrein-5 | |
| inhibitor: | BDBM32834 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Materials Human kallikrein 5 (hK5) was purchased from R&D Systems (Cat #1108-SE). Substrate Boc-Val-Pro-Arg-AMC was from Bachem (Cat #I-1120.0050). Assay buffer consisted of 50 mM Tris, pH 8.0, 0.05% Tween 20. 1536-well black plates were from Corning (Item #3728). Assay hK5 (0.60 ug/mL) was incubated with Boc-Phe-Ser-Arg-AMC substrate (15 uM) in 5 uL of assay buffer (see above) for 2 hr at room temperature. HTS was performed using 5 uM compound. 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 2 transfers of 100 nL with the Evolution 384 pintool (washed with isopropanol after each transfer) 5.Add 1 uL of Boc-Val-Pro-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro 6.Add 5 uL enzyme (1.35 ug/mL in assay buffer) usi | |
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