| Assay Method Information | |
| | Rapid-Fire Mass Spectrometry (RFMS) Assay |
| Description: | A rapid-fire mass spectrometry (RFMS) assay was used to assess RNR enzyme activity using a 384 well plate and a robotic platform. The plate layout included two validated reference compounds (Triapine (3-AP) and Hydroxyurea (HU)): A dose response in duplicate; top concentration: 5 uM (3-AP) and 250 uM (HU), semi-log dilutions. Spike wells in triplicate randomly spotted at four concentrations: 250 uM, 100 uM, 30 uM and 2 uM for HU 5 uM, 2 uM, 0.6 uM and 0.04 uM for 3-AP First, the multidrop pipes were saturated for 30 minutes with enzymatic solution. Then 30 uL of Stop solution was distributed in column 24. Next, 15 uL 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 uL of substrate solution (column 1 to 24). Next, the plate was incubated for 45 minutes at 37 C. 30 uL 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 uM; [ATP]: 1 mM; [NADPH]: No [RNR]final: 50 nM with 1:1 (RNR1:RNR2) ratio Final volume: 30 uL Stop solution: 6% HCOOH containing 2 uM of 15 The compounds were screened at concentrations from 10 nM to 30 uM. |
| Affinity data for this assay | |
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