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| | AMPD Enzymatic Activity Assay |
| Description: | AMPD1 was added to buffer A (50 mM HEPES, 150 mM KCl, 5 mM MgCl2, and 0.5 mMglutathione [pH 7.4]) to a concentration of 10 nM. A substrate mix consistingof 2mMNADPH, 6mMa-ketoglutarate, 8mMAMP, and 15 U GDH was prepared in buffer A. Substrate mix (4 ml) was added to the plate, followed by centrifugation and incubation for 1 hr. Detection was by absorbance at 340 nm following the conversion of NADPH to NADP+. Recombinant AMPD enzyme activity assays were performed in a 384-well format using the same conditions as described above, except the assay volume was 80 ml and the AMP concentration for the AMPD2 assay was 8 mM. Different AMPD enzyme concentrations were used to have linear reactions: hAMPD1 (4 nM), hAMPD2 (38 nM), hAMPD3 (1 nM), rAMPD1 (116 nM), rAMPD2 (53 nM), rAMPD3 (31 nM), and mAMPD1 (816 nM). |
| Affinity data for this assay | |
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