| Assay Method Information | |
| | RNR Enzyme Activity Assay |
| Description: | A rapid-fire mass spectrometry (RF/MS) assay was used to assess RNR enzyme activity using a 384 well plate and a robotic platform.[0402]The plate layout included two validated reference compounds (Triapine (3-AP) and Hydroxyurea (HU)):A dose response in duplicate; top concentration: 5 μM (3-AP) and 250 μM (HU), semi-logdilutions.Spike wells in triplicate randomly spotted at four concentrations:250 μM, 100 μM, 30 μM and 2 μM for HU5 μM, 2 μM, 0.6 μM and 0.04 μM for 3-APFirst, the multidrop pipes were saturated for 30 minutes with enzymatic solution. Then 30 μL of Stop solution was distributed in column 24. Next, 15 μL of enzyme was distributed in column 1 to 24. Next, a pre-incubation step of 15 minutes at room temperature occurred, followed by distribution of 15 μL of substrate solution (column 1 to 24). Next, the plate was incubated for 45 minutes at 37° C. 30 μL of Stop solution was distributed to columns 1 to 23.The final parameters for the enzyme reactions were:Incubation: 37° C., 45 min[CDP]: 5 μM; [ATP]: 1 mM; [NADPH]: No[RNR]final: 50 nM with 1:1 (RNR1:RNR2) ratioFinal volume: 30 μLStop solution: 6% HCOOH containing 2 μM of 15 |
| Affinity data for this assay | |
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