For ATP consumption, 16 μmol/L xtFAM46B was incubated with 80 μmol/L A15 primer and 80 μmol/L ATP in a buffer containing 20 mmol/L HEPES pH 7.5, 150 mmol/L NaCl, 2 mmol/L MgCl2, and 2 mmol/L KCl at 37°C for 20 min. The mixture was then analyzed by an HPLC system (Agilent, Santa Clara, CA, USA) equipped with a reverse phase C18 ODS‐2 Hypersil analytical column preceded by a C18 guard column (Thermo Scientific), with 100 mmol/L potassium phosphate pH 6.5, 10 mmol/L tetrabutyl ammonium bromide, and 7.5% acetonitrile as running buffer. ATP was detected by absorption at 254 nm and quantified by integration of the corresponding peaks.
C18 ods 2 hypersil analytical column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The C18 ODS-2 Hypersil analytical column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. The column features a silica-based stationary phase with octadecylsilane (C18) functional groups, providing a reversed-phase separation mechanism. The Hypersil packing material offers efficient and reproducible chromatographic performance.
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3 protocols using c18 ods 2 hypersil analytical column
1
Quantitative ATP Consumption Assay
2
Kinetic Analysis of RNA Synthesis
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A total of 500 nM protein was incubated with 2.5 μM RNA primer and 25 μM NTP in a buffer containing 20 mM Tris–HCl pH 8.0, 300 mM NaCl, 2 mM MgCl2 and 2 mM DTT at 37°C. At each time point, 10 μl sample was collected and prepared for subsequent analysis with a final volume of 25 μl. The HPLC system (Agilent) was equipped with a reverse phase C18 ODS-2 Hypersil analytical column preceded by a C18 guard column (Thermo Scientific), with 100 mM potassium phosphate pH 6.5, 10 mM tetrabutyl ammonium bromide and 10% acetonitrile as running buffer. Nucleotides were detected by absorption at 256 nm and quantified by integration of the corresponding peaks.
3
In vitro RNA synthesis assay
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16 μM protein was incubated with 80 μM A15 primer and 80 μM NTP in a buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 4 mM MgCl2 and 4 mM KCl at 37°C for 20 min. The mixture was then analyzed by an HPLC system (Agilent) equipped with a reverse phase C18 ODS-2 Hypersil analytical column preceded by a C18 guard column (Thermo Scientific), with 100 mM potassium phosphate pH 6.5, 10 mM tetrabutyl ammonium bromide and 7.5% acetonitrile as running buffer. Nucleotides were detected by absorption at 254 nm and quantified by integration of the corresponding peaks.
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