| Assay Method Information | |
| | CD73 Enzyme Inhibition Assay |
| Description: | Test compound solution preparation: Stock solution of a test compound was prepared in DMSO at 10 mM concentration. A series of preset 10-concentration solutions of a test compound were prepared by diluting the stock solution in 5-fold gradient dilution with DMSO. Assay buffer (lx): 20 mm Tris, 25 mM NaCl, 1 mM MgCl2, pH 7.5, 0.005% Tween-20 (freshly prepared, and ready for use). An aliquot of 0.25 uL of compound solution or DMSO (blank control) was added to each test well of a micro well plate. Recombinant human 5'-nucleotidase (hCD73) was diluted to 1 nM in assay buffer; and an aliquot of 25 uL hCD73 (1 nM) solution was added to the corresponding wells of the plate. The contents of the well were well-mixed, and the plate was covered with sealing film, centrifuged at 1000 rpm for 30 sec., followed by incubation at room temperature for 15 min. AMP, the substrate, was diluted to 60 uM concentration in the assay buffer. To each reaction well was added 25 uL of AMP solution. After mixing well, the plate was covered with sealing film, centrifuged at 1000 rpm for 30 sec., and incubated at 37 C. for 20 min. The absorbance signal (end point) was measured on SPARK plate reader at 635 nm (See PiColorLock Gold Phosphate Detection System (Abcam) test kit for reference). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |