| Assay Method Information | |
| | Ecto-5'-Nucleotidase Inhibition Assay |
| Description: | The enzymatic assay of both human and rat ecto-5'-nucleotidase were performed with slight modifications in the previously described method. The stock solution of compounds were prepared with concentration of 10mM in 10% DMSO and their activity were determined by diluting them further in assay buffer (1 mM CaCl2, 2 mM MgCl2 and 10 mM Tris HCl (pH 7.4) to 1mM concentration. The final assay volume 100 μL contained 70 μL of assay buffer (2 mM MgCl2, 1 mM CaCl2 and 10 mM Tris HCl, pH 7.4), 10 μL of compound and 10 μL of human e5'NT (6.94 μg/mL) protein extract or rat e5'NT (7.17 μg/mL). Then the reaction mixture was incubated for 10 min at 37 °C. Afterwards, 10 μL of AMP substrate was added to initiate the reaction. The reaction was stopped by providing temperature of 99 °C for 20 min. Aliquots of 50 μL of each reaction mixture was transferred into CE mini vial and hydrodynamically injected into the capillary by using pressure of 0.5 psi for 5s. The electrosmatic separation of substrate and product peaks occurred by applying voltage of 15 kV. The concentration of product i.e. adenosine was estimated by calculating the area under its absorbance peak at 260 nm. |
| Affinity data for this assay | |
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