Assay Method Information

Assay Name:  Alkaline Phosphatase Inhibition Assay
Description:  Alkaline phosphatase (b-TNAP, c-IAP purchased from Calzyme Laboratories, Inc. USA) inhibition assay of different cyclic sulfonamides was performed using a chemiluminescent substrate i.e. CDP-Star® (disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2'-(5-chlorotricyclo[]decan])-4-yl]-1-phenyl phosphate). With a slight modification in reported spectrophotometric procedure, an optimized conditions of the bioassay were developed [Hessle et al., Proc. Natl. Acad. Sci., 99:9445-9449]. Buffer solution for this bioassay was composed of 8 M DEA (pH 9.8) that contain 2.5 mM MgCl2 and 0.05 mM ZnCl2. First of all, the compounds were tested at the final concentration of 200 μM(with final DMSO 1% (v/v)). In bioassay procedure, the total volume (50 μL solution) contained 10 μL of tested compound, followed by the addition of 20 μL of TNAP (1:800 times diluted (0.8 unit/mL) enzyme in assay buffer) or 20 μL of IAP (1:800 times diluted (1 unit/mL) enzyme in assay buffer). The mixture was allowed to incubate for 3-5 min at 37 °C and luminescence was measured using microplate reader (BioTek FLx800, Instruments,Inc. USA). The reaction was initiated by the addition of 20 μL of CDP-star® (final concentration of 110 μM) and the assay mixture was incubated again at 37 °C. The change in the luminescence was measured after for 15 min of incubation. The activity of each compound was compared with total activity control (without any inhibitor). Levamisole (2 mM per well) and L-phenylalanine (4 mM per well) were used as a standard inhibitors for tissuenonspecific alkaline phosphatase (TNAP) and calf intestinal alkaline phosphatase (IAP), respectively.
Affinity data for this assay

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