| Solution Information | help | |
| Enzyme: | Furin | |
| inhibitor: | BDBM75780 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Biochemical Furin HTS Assay protocol Reaction Buffer Concentrations (final concentrations): 50 mM Hepes pH 7.5, 1 mM CaCl2, 1 mM beta-mercaptoethanol , 0.2 mg/ml BSA. Substrate Stock Solution: 10 mM pERTKR-AMC in DMSO; stored in aliquots at -20 oC. rhFurin714 prep (5.2 units/uL)Stored in aliquots at -80oC. Furin Assay Protocol The assay involves three liquid transfer steps of 5 uL each of 30 uM compound in 1% DMSO (10 uM final), 1 unit/5 uL rhFurin714 in 3X reaction buffer and 5 #L of 30 uM pERTKR-AMC substrate (10 uM final). The assay plates used are Greiner 384-well, flat-bottom, low volume, black polystyrene plates (VWR catalog # 784076). 1. Add 5 uL of compound/controls to each well. 2. Add 5 uL of rhFurin714 in 3X buffer (1.0 units/well final). 3. Add 5 uL of 30 uM pERTKR-AMC fluorigenic substrate (10 uM/well final). 4. Incubate the plates for 1 hr at room temperature. 5. Stop the reaction by adding 5 uL of 1M Acetic acid. 6. Measure the amount of fluorigenic AMC | |
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