| Assay Method Information | |
| | Test Method for CD73 Enzyme Activity In Vitro |
| Description: | Human recombinant protein CD73 (Acro, Cat. No. CD3-H52H7) was prepared into a 0.04 ng/μL protein solution, and substrate AMP (Sigma, Cat. No. A1752-1G) was prepared into a 100 μM substrate solution. 5 μL of compounds at different concentrations were added into a 384-well white transparent bottom plate, 5 μL of 0.04 ng/μL recombinant CD73 enzyme solution was added, and the mixture was incubated at room temperature for 15 minutes. 10 μL of 100 μM substrate solution was added, the mixture was incubated at 37° C. for 40 minutes, 5 μL of Malachite Green Reagent A (R&D, Cat. No. DY996) was added, the mixture was incubated at room temperature for 10 minutes, and finally, 5 μL of Malachite Green Reagent B was added, the mixture was incubated at room temperature for 20 minutes, and a microplate reader PHERAstar FSX instrument was used to detect the absorbance at 620 nm. IC50 values were calculated using the origin9.2 software. |
| Affinity data for this assay | |
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