1450 microbeta micoplate scintillation and luminescence counter

Aminoacylation reactions were carried out in an assay solution containing 50 mM HEPES pH 7.5, 60 mM KCl, 10 mM MgCl₂, 4 mM ATP, 5 mM DTT, 4 μg/mL yeast pyrophosphatase (Roche), 1 mM Spermine, 10 μM cold L-serine, and 5 μM [³H]-serine (1 mCi/mL). Varying amounts of tRNA were initially mixed with assay solution, and the reaction was initiated by addition of human mSerRS (0.5 µM). At varying time intervals, 5 μL aliquots were removed and applied to a MultiScreen 96-well filter plate (0.45 μm pore size hydrophobic, low-protein-binding membrane; Merck Millipore), pre-wetted with quench solution (0.5 mg/mL salmon sperm DNA, 0.1 M EDTA, 0.3 M NaOAc (pH 3.0)). After all time points were collected, 100 μL of 20% (w/v) trichloroacetic acid (TCA) was added to precipitate nucleic acids. The plate was then washed four times with 200 μL of 5% TCA containing 100 mM cold serine, followed once by 200 μL of 95% ethanol. The plate was then dried, followed by deacylation of bound tRNAs by addition of 70 μL of 100 mM NaOH. After 10 min incubation at RT, the NaOH-solution was centrifuged into a 96-well flexible PET microplate (PerkinElmer) with 150 μL of Supermix scintillation mixture (PerkinElmer). After mixing, the radioactivity in each well of the plate was measured in a 1450 MicroBeta Micoplate Scintillation and Luminescence Counter (PerkinElmer).

Aminoacylation reactions were carried out in an assay solution containing 50 mM HEPES pH 7.5, 60 mM KCl, 10 mM MgCl₂, 4 mM ATP, 5 mM DTT, 4 μg/mL yeast pyrophosphatase (Roche), 1 mM Spermine, 10 μM cold L-serine, and 5 μM [³H]-serine (1 mCi/mL). Varying amounts of tRNA were initially mixed with assay solution, and the reaction was initiated by addition of human mSerRS (0.5 µM). At varying time intervals, 5-μL aliquots were removed and applied to a MultiScreen 96-well filter plate (0.45 μm pore size hydrophobic, low-protein-binding membrane; Merck Millipore), pre-wetted with quench solution (0.5 mg/mL salmon sperm DNA, 0.1 M EDTA, 0.3 M NaOAc (pH 3.0)). After all time points were collected, 100 μL of 20% (w/v) trichloroacetic acid (TCA) was added to precipitate nucleic acids. The plate was then washed four times with 200 μL of 5% TCA containing 100 mM cold serine, followed once by 200 μL of 95% ethanol. The plate was then dried, followed by deacylation of bound tRNAs by addition of 70 μL of 100 mM NaOH. After 10 min incubation at RT, the NaOH-solution was centrifuged into a 96-well flexible PET microplate (PerkinElmer) with 150 μL of Supermix scintillation mixture (PerkinElmer). After mixing, the radioactivity in each well of the plate was measured in a 1450 MicroBeta Micoplate Scintillation and Luminescence Counter (PerkinElmer).

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.).