Pvdf multiscreen filter plates

The concentration of active sites was determined at RT (25 °C) in 40 μl reactions containing two different concentrations (5 and 10 μM) of human mSerRS, 20 mM L-serine, 22 nM [γ-³²P]-ATP, in assay buffer (100 mM HEPES pH 7.5, 20 mM KCl, 10 mM MgCl₂, 2 mM DTT, and 2 mg/mL yeast pyrophosphatase (Roche)). Reactions were initiated by adding enzyme to the assay solution in 96-well low-profile PCR plates. At different time points 5 μl reaction mix were quenched into PVDF MultiScreen filter plates (0.45 μm pore size hydrophobic, low-protein-binding membrane; Merck Millipore) containing 20 μl of 7% HClO₄ and 80 μl of 10% charcoal slurry. Following the last time point, the slurry was mixed by pipetting and centrifuged into a 96-well flexible PET microplate (PerkinElmer) containing 150 μL of Supermix scintillation mixture (PerkinElmer). The plate was counted on a 1450 MicroBeta Micoplate Scintillation and Luminescence Counter (PerkinElmer). Kinetic data were analyzed using GraphPad Prism 8 (GraphPad Software, Inc.).

The concentration of active sites was determined at RT (25 °C) in 40-μl reactions containing two different concentrations (5 and 10 μM) of human mSerRS, 20 mM L-serine, 22 nM [γ-³²P]-ATP, in assay buffer (100 mM HEPES pH 7.5, 20 mM KCl, 10 mM MgCl₂, 2 mM DTT, and 2 mg/mL yeast pyrophosphatase (Roche)). Reactions were initiated by adding enzyme to the assay solution in 96-well low-profile PCR plates. At different time points 5 μl reaction mix were quenched into PVDF MultiScreen filter plates (0.45 μm pore size hydrophobic, low-protein-binding membrane; Merck Millipore) containing 20 μl of 7% HClO₄ and 80 μl of 10% charcoal slurry. Following the last time point, the slurry was mixed by pipetting and centrifuged into a 96-well flexible PET microplate (PerkinElmer) containing 150 μL of Supermix scintillation mixture (PerkinElmer). The plate was counted on a 1450 MicroBeta Micoplate Scintillation and Luminescence Counter (PerkinElmer). Kinetic data were analyzed using GraphPad Prism 8 (GraphPad Software, Inc.).

The concentration of active sites was determined at room temperature (25 °C) in 40-μl reactions containing two different concentrations (5 and 10 μM) of Hs mt aaRS (AlaRS or SerRS), 20 mM L-amino acid, 22 nM [γ-³²P]-ATP, in assay buffer (100 mM Hepes pH 7.5, 20 mM KCl, 10 mM MgCl₂, 2 mM DTT, and 2 mg/mL pyrophosphatase). Reactions were initiated by adding enzyme to the assay solution in 96-well low-profile PCR plates. At different time points 5 μl reaction mix were quenched into PVDF MultiScreen filter plates (0.45 μm pore size hydrophobic, low-protein-binding membrane; Merck Millipore) containing 20 μl of 7% HClO₄ and 80 μl of 10% charcoal slurry. Following the last time point, the slurry was mixed by pipetting and centrifuged into a 96-well flexible PET microplate (PerkinElmer) containing 150 μL of Supermix scintillation mixture (PerkinElmer). The plate was counted on a 1450 MicroBeta Micoplate Scintillation and Luminescence Counter (PerkinElmer).