Up to 1 μg RNA in 30 μl aqueous digestion mix were digested to single nucleosides by using 0.2 u Alkaline Phosphatase, 0.02 u Phosphodiesterase I (VWR, Radnor, PA, USA) and 0.2 u Benzonase in 5 mM TRIS (pH 8.0) and 1 mM MgCl2. Furthermore, 0.5 μg tetrahydrouridine (Merck, Darmstadt, Germany), 1 μM butylated hydroxytoluene and 0.1 μg pentostatine were added. The mixture was incubated with the RNA for 2 h at 37°C and filtered through 96-well filterplates (AcroPrep™ Advance 350 10 K Omega™, PALL Corporation, New York, USA) at 4°C for 30 min at 3000 × g, or through single tubes (VWR, Partnumber: 516-0229) at room temperature for 7 min at 5000 × g. The filtrate was mixed with 1/10 Vol. of 10× yeast SILIS (stable isotope labeled internal standard) (38 ) for absolute quantification.
Alkaline phosphatase
Manufactured by Avantor
Sourced in United States
Alkaline phosphatase is an enzyme that catalyzes the hydrolysis of phosphate esters in an alkaline environment. It is commonly used in various laboratory applications as a marker for certain biological processes.
Automatically generated - may contain errors
5 protocols using alkaline phosphatase
1
RNA to Nucleosides Conversion Protocol
2
tRNA Nucleoside Quantification Protocol
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tRNA (100 ng) in aqueous digestion mix (30 μL) was digested to single nucleosides by using 0.2 U alkaline phosphatase, 0.02 U phosphodiesterase I (VWR, Radnor, Pennsylvania, USA), and 0.2 U benzonase in Tris (pH 8, 5 mM) and MgCl2 (1 mM) containing buffer. Furthermore, 0.5 µg tetrahydrouridine (Merck, Darmstadt, Germany), 1 µM butylated hydroxytoluene, and 0.1 µg pentostatin were added to avoid deamination and oxidation of the nucleosides30 (link). The mixture was incubated for 2 h at 37 °C and then filtered through 96-well filter plates (AcroPrep Advance 350 10 K Omega, PALL Corporation, New York, USA) at 3000 × g and 4 °C for 30 min. 1/10 Vol. of SILIS (stable isotope labeled internal standard) as prepared in31 (link) was added to each filtrate before analysis by QQQ mass spectrometry.
3
Comprehensive RNA Nucleoside Analysis
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Total tRNA (300 ng), 18S or 28S rRNA (500 ng each) or Poly-A RNA (100 ng) were digested in aqueous digestion mix (30 μL) to single nucleosides by using 2 U alkaline phosphatase, 0.2 U phosphodiesterase I (VWR, Radnor, Pennsylvania, USA), and 2 U benzonase in Tris (pH 8, 5 mM) and MgCl2 (1 mM) containing buffer. Furthermore, 0.5 μg tetrahydrouridine (Merck, Darmstadt, Germany), 1 μM butylated hydroxytoluene, and 0.1 μg pentostatin were added. After incubation for 2 h at 37 °C, 20 μL of LC-MS buffer A (QQQ) was added to the mixture and then filtered through 96-well filter plates (AcroPrep Advance 350 10 K Omega, PALL Corporation, New York, USA) at 3000 × g at 4 °C for 30 min. A stable isotope labelled SILIS (gen2,27 (link)) was added to each filtrate and calibration solution of synthetic standards before injection into the QQQ MS.
4
Nucleoside Analysis of tRNA by LC-MS
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The Trichoplusia ni tRNA that copurified with the SerRS-METTL6 fusion construct was isolated via chloroform-phenol extraction and precipitated with ethanol and sodium acetate. Around 300 ng of the tRNA was digested in an aqueous digestion mix (30 μl) to single nucleosides by using 2 U alkaline phosphatase, 0.2 U phosphodiesterase I (VWR) and 2 U benzonase in Tris (pH 8, 5 mM) and MgCl 2 (1 mM) containing buffer. Furthermore, 0.5 μg tetrahydrouridine (Merck), 1 μM butylated hydroxytoluene and 0.1 μg pentostatin were added. After incubation for 2 h at 37 °C, 20 μl of LC-MS buffer A (QQQ) was added to the mixture. A stable isotope labeled SILIS (gen² (ref. 79)), was added to each replicate and calibration solution of synthetic standards before injection into the QQQ MS.
5
Quantitative Analysis of tRNA Nucleosides
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Total tRNA (300 ng) in aqueous digestion mix (30 μL) was digested to single nucleosides by using 2 U alkaline phosphatase, 0.2 U phosphodiesterase I (VWR, Radnor, Pennsylvania, USA), and 2 U benzonase in Tris (pH 8, 5 mM) and MgCl2 (1 mM) containing buffer. Furthermore, 0.5 µg tetrahydrouridine (Merck, Darmstadt, Germany), 1 µM butylated hydroxytoluene, and 0.1 µg pentostatin were added to avoid deamination and oxidation of the nucleosides. When quantification of dihydrouridine was intended tetrahydrouridine was omitted. After incubation for 2 h at 37 °C, 20 µL of LC-MS buffer A (QQQ) was added to the mixture and then filtered through 96-well filter plates (AcroPrep Advance 350 10 K Omega, PALL Corporation, New York, USA) at 3000 × g and 4 °C for 30 min. A stable isotope labeled internal standard (SILIS) was produced in S. cerevisiae using 13C and 15N rich growth medium (Silantes, Munich, Germany, Product# 111601402) following recently described procedures16 ,31 (link). 1/10 Vol. of SILIS was added to each filtrate before analysis by QQQ mass spectrometry. For each sample 10 µL were injected (~60 ng of sample tRNA).
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