Indicated ATPase reactions were set-up as described above in microcentrifuge tubes. After completion of reactions, each tube was centrifuged at 10,000 x g for 2 minutes, and then 15 μL of supernatant was loaded onto a 96 well plate (Thermo Scientific), diluted with 35 μL of 50 mM potassium phosphate (pH 6.0) as running buffer. HPLC analysis was conducted with an autosampler using a 2.6 μm, 4.6 x 150 mm Accucore aQ C18 Polar Endcapped column (ThermoFisher Scientific). Each sample (15 μL) was eluted with an isocratic gradient of running buffer at a flow rate of 0.700 mL/min for 10 min at a column temperature of 30 °C. The presence of ATP and ADP was detected at a wavelength of 260 nm and quantified using Chromeleon 7 software (ThermoFisher Scientific) using ATP/ADP standards that were analyzed under the same conditions.
Accucore aq c18 polar endcapped column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Accucore aQ C18 Polar Endcapped column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of polar and non-polar analytes. The column features a C18 stationary phase with a polar endcapping, providing enhanced retention and selectivity for polar compounds.
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2 protocols using accucore aq c18 polar endcapped column
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HPLC-based Quantification of ATP and ADP
2
Targeted Metabolite Profiling by UHPLC-HRMS
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Chromatographic column: Accucore™ aQ C18 Polar Endcapped column, 150 × 2.1 mm, 2.6 µm (Thermos Fisher Scientific, Waltham, MA, USA); column temperature: 30 °C; flow rate: 0.3 mL/min; samples injection volume: 5.0 μL; mobile phase A: water with 0.1% (v/v) acetic acid; mobile phase B: acetonitrile with 0.1% (v/v) acetic acid. The column was eluted with gradient mobile phase B: it was kept at 2% B for 2 min, increased to 50% B after 5 min, then increased to 100% after 10 min and kept at 100% for 2 min. Before the next injection, the column was equilibrated at 2% B for 5 min.
The eluted metabolites were detected separately by positive and negative ion modes, with the Full MS-AIF method on a Q Extractive Focus instrument (Thermos Fisher Scientific, Waltham, MA, USA). Full MS resolution: 70,000; scan range: 70~1000 m/z; AGC target: 1.0 × 106; spectrum data type: profile; AIF resolution: 70,000; AIF scan range: 70~1000 m/z; stepped NCE (normalized collision energy): 10 V, 20 V and 40 V; AIF spectrum data type: profile.
The eluted metabolites were detected separately by positive and negative ion modes, with the Full MS-AIF method on a Q Extractive Focus instrument (Thermos Fisher Scientific, Waltham, MA, USA). Full MS resolution: 70,000; scan range: 70~1000 m/z; AGC target: 1.0 × 106; spectrum data type: profile; AIF resolution: 70,000; AIF scan range: 70~1000 m/z; stepped NCE (normalized collision energy): 10 V, 20 V and 40 V; AIF spectrum data type: profile.
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