| Assay Method Information | |
| | CD73 Biochemical IC50 Assay |
| Description: | Compound serial dilutions were pre-spotted into Thermo Nunc assay plate. 50 uL CD73 enzyme buffer (CD73 purchased from R&D system=0.6 nM, 25 mM Tris, pH 7.4, 5 mM MgCl2, 1 mM NaH2PO4) was added into the assay plate and incubate for 15 mins. 50 uL AMP buffer (AMP=30 uM in 25 mM Tris, pH 7.4, 5 mM MgCl2, final AMP=15 uM, 2×Km, final CD73=0.3 nM) was added into assay plate. After incubation for 60 mins, the supernatant (20 uL) was transferred into 384-well NUNC plate pre-filled with 60 uL Quench buffer (80% organic and 20% water+0.1% FA) with internal standard. The plate was spun down at 4500 rpm for 20 mins, then 20 uL of supernatant was transferred to another Nunc plate prefilled with 80 uL of water. The samples were analyzed using Rapid fire. |
| Affinity data for this assay | |
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