| Assay Method Information | |
| | Biochemical CD73 Assay |
| Description: | Activity of recombinant CD73 was measured by quantification of free phosphate using the Malachite Green detection system (R&D Systems #DY996). Test compounds were solubilized in DMSO and dispensed into 384-well polystyrene plates in a 8-point 3× titration in duplicates. Then, 10 μL of 4 nM human CD73 enzyme (Novoprotein #C446) in phosphate-free assay buffer (10 mM HEPES, pH 7.4, 125 mM NaCl, 1 mM KCl, 10 mM glucose, 2 mM MgCl2) was added to the plates. The compound and enzyme were incubated for 15 min at RT. Then, 10 μL of 80 μM AMP (Sigma Aldrich #A1752) in the assay buffer was added and the reaction mixture was incubated for 45 min at 37° C. The final concentrations of CD73 and AMP in the reaction were 2 nM and 40 μM, respectively. The reaction was stopped by adding 5 μL of Malachite Green reagent A and incubating for 10 min at RT, followed by addition of 5 μL of Malachite Green reagent B and incubation for 20 min at RT. Absorbance was then read at 620 nM with the Flexstation spectrophotometer (Molecular Devices). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |