| Assay Method Information | |
| | CD73 Inhibition Assay |
| Description: | Compound inhibition of human (h)CD73 (amino acids Trp27-Lys547, R&D Systems 5795-EN-500) enzymatic activity was tested via the Malachite Green Phosphate Detection Kit (R&D DY996). Experiments were performed in 20 μL Tris buffer (25 mM Tris pH 7.5, 5 mM MgCl2, 0.002% Tween20), with 250 pM of hCD73. Test compounds of interest were prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration, with the assay DMSO concentration being 1%. Compounds were pre-incubated for 20 minutes with hCD73 in a 384 well clear plate (Greiner 781101), followed by the addition of adenosine monophosphate (AMP) to 12.5 μM. The enzymatic reaction was carried out for 20 min at room temperature. 5 μL of Malachite kit reagent A was added to stop the reaction (contains 3M H2SO4) and after 10 min, 5 μL of Malachite kit reagent B was added (Malachite Green). After 30 min, absorbance at 620 nm was read on a BMG Pherastar FSX. |
| Affinity data for this assay | |
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