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| | Alkaline Phosphatase Inhibition Assay |
| Description: | The assay conditions were optimized with some changes in formerly reported spectrophotometric method. The composition of assay buffer of pH 9.8 was as: 8 M diethanolamine (DEA), 0.05 mM ZnCl2 and 2.5 mM MgCl2. All compounds were tested at the initial concentration of 0.2 mM. A total assay volume of 50 μL contained 10 μL of tested compound, followed by the addition of 20 μL of either b-TNAP (1:800 times diluted enzyme in assay buffer) or 20 μL of c-IAP (1:800 times diluted enzyme in same assay buffer). After incubating the reaction mixture for 3-5 minutes at 37 °C, pre-read was taken by measuring luminescence with the help of microplate reader (BioTek FLx800, Instruments, Inc. USA). After that, 20 μL of substrate (CDP-Star) at the concentration of (110 μM) was added to the reaction mixture. Then the reaction mixture was again incubated for 15-20 min at 37 °C and after read was taken to measure the change in the luminescence. Total activity control (without inhibitor or negative control) was used for comparison of the activity of each test compound. L-phenylalanine (at concentration of 4 mM per well) and Levamisole (at concentration of 2 mM per well) were used as reference standards against calf intestinal alkaline phosphatase (c-IAP) and bovine tissue-nonspecific alkaline phosphatase (b-TNAP) respectively. |
| Affinity data for this assay | |
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