| Assay Method Information | |
| | In Vitro Inhibitory Activity of Compound to Recombinant Human CD73 Enzyme |
| Description: | The test was performed using Tris-MgCl2 buffer containing 25 mM Tris (Biosharp; 77-86-1) and 25 mM MgCl2 (Nanjing Chemical Reagent Co., Ltd.; 7791-18-6). Human-CD73 (Novoprotein; C446) was diluted with Tris-MgCl2 buffer to form a stock solution (3×), which was placed in a 96-well plate according to 20 μL/well to obtain a final concentration of 0.1 μg/mL. The compound was diluted with Tris-MgCl2 buffer to form a stock solution (3×) of an appropriate concentration gradient, the compound solution was added to the above 96-well plate according to 20 μL/well and uniformly mixed with the Human-CD73 solution, and the mixture was incubated at the room temperature for 30 min. Meanwhile, a positive reference group (without the compound) and a negative reference group (without CD73) were set. AMP (Sigma; A1752-5G) was diluted with Tris-MgCl2 buffer to form a stock solution (3×), which was added to the above 96-well plate according to 20 μL/well to obtain a final concentration of 100 μM, and the mixture was uniformly mixed and incubated at 37° C. for 60 min; ATP (Sigma; A7699-1G) was diluted with Tris-MgCl2 buffer to form a stock solution (7×), which was added to the above 96-well plate according to 10 μL/well to obtain a final concentration of 100 μM, and the mixture was uniformly mixed, incubated for 5 min, and tested by using an ATP-GLO kit (Promega; G7573). |
| Affinity data for this assay | |
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