| Assay Method Information | |
| | DEN2V Protease Inhibition Assay |
| Description: | The enzyme inhibition assay was performed in 384-well flat bottom black polystyrene microplates (cat. N. 781900, Greiner,) in a reaction volume of 20 μl. DEN2V NS2B-G4SG4-NS3 serine protease (MGSSHI-HHIHHHSSGLVPRGSHMADLELERAADVRWEEQAEISGSSPILSITISED GSMSIKNEEEEQTLGGGGSGGGGAGVLWDVPSPPPVGKAELEDGAYRIKQKGILGYSQI GAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKE GEEVQVLALEPGKNPRAVQTKPGLFKTNTGTIGAVSLDFSPGTSGSPIVDKKGKVVGLY GNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRK; SEQ ID No 2) (10 nM) was incubated with increasing inhibitor concentrations (0.097-100 μM or 0.0009-1 μM for the most active compounds) in assay buffer (50 mM Tris-HCl, pH 8.5, 1% glycerol, 1 mM CHAPS, 1% DMSO) for 10 minutes at 25° C. 15 μM of Bz-Nle-KRR-AMC substrate (cat. N. 4055312, Bachem) was added and the reaction mixture incubated for additional 30 minutes at 25° C. Product formation was evaluated by fluorescence measurement (excitation 360 nm, emission 465 nM), using a SPARK TM10 Tecan instrument. Results were analyzed using Prism (GraphPad, San Diego, CA) and Vortex software (Dotmatics, Bioshops Stortford, UK). Dose-response curves were fitted by four-parameter logistic regression. |
| Affinity data for this assay | |
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