| Assay Method Information | |
| | Ecto-5'-Nucleotidase Inhibition Assay |
| Description: | The ecto-5'-nucleotidase inhibition assay was performed according to our previously reported protocol [Raza et al., Med. Chem., 8:1133-1139]. The compounds were analyzed at final concentration of 0.1 mM prepared in assay buffer ((2 mM MgCl2, 10 mM Tris HCl and 1 mM CaCl2, pH 7.4). (2 mM MgCl2, 10 mM Tris HCl and 1 mM CaCl2, pH 7.4). The total 100 μL containing 10 μL of sample, 10 μL of h-e5'NT (6.94 μg/mL) protein extract or r-e5'NT (7.17 μg/mL) and 70 μL of assay buffer. Then the reaction mixture was allowed to incubate for 10 min at 37 °C. After incubation, 10 μL of substrate AMP (adenosine monophosphate) with final concentration 500 μM was added to start the enzymatic reaction. The mixture was again allowed to incubate for 30 min at 37 °C. Then the mixture was placed in water bath at 99 °C for 20 min to stop the enzymatic reaction by thermal denaturation of protein. The aliquots of 50 μL of this reaction mixture were transferred into CE mini vials and injected hydrodynamically into the capillary under pressure of 0.5 psi for 5 s, followed by the application of 15 kV voltage for separation of substrate and product peaks. |
| Affinity data for this assay | |
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