Assay Method Information

Assay Name:  Alkaline Phosphatase Assay
Description:  Alkaline phosphatase assay was optimized and performed in the same way as previously reported method with slight modifications [Sergienko et al., Nat. Protoc., 5:1431-1439]. All compounds were analyzed at a final concentration of 200 μM (1% DMSO) by using assay buffer with composition of 8 M diethanolamine (DEA) (pH 9.8), 0.05 mM ZnCl2 and 2.5 mM MgCl2. The total reaction volume was kept 50 μL containing 10 μL of tested compound, 20 μL of b-TNAP (1:800 (0.8 units/mL) diluted enzyme in assay buffer) or c-IAP (1:800 (1 units/mL) diluted enzyme in assay buffer). After incubation for 3-5 min at 37 °C., the luminescence was measured as a pre-read using microplate reader (BioTek FLx800, Instruments, Inc. USA). Then, 20 μL of substrate (CDPstar®) (final concentration of 110 μM) was added to the reaction mixture to initiate the enzymatic reaction. The change in the luminescence was recorded after 15 min. at 37 °C. The activity of each compound was measured in comparison with control (without any inhibitor). Levamisole (2 mM per well) and L-phenylalanine (4 mM per well) were used as standards (positive control) against b-TNAP) and c-IAP), respectively.
Affinity data for this assay

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