The analysis of AF samples was carried out using nuclear magnetic resonance (NMR) spectroscopy (Figure 1 ) following a previously published protocol by our group [26 (link)].
For the AF sample preparation, 400 μL D2O and 150 μL phosphate buffer in D2O were added to lyophilized AF samples. After centrifugation (4500× g, 15 °C, 5 min), 50 μL of sodium maleate was added as an internal standard to 500 μL of the supernatant, and the sample was transferred to 5 mm NMR tubes.
Sodium maleate was chosen as the reference standard since it is suitable for the CPMG pulse sequence and provides a distinct peak in the 1 H NMR spectrum. The samples were thawed at room temperature for 60 min before performing NMR experiments.
All NMR spectra were acquired using a Varian-600MHz NMR spectrometer equipped with a triple resonance probe {HCN} at 25 °C. The Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence was applied to AF samples with 128 transients collected with 64 K data points. The relaxation delay was set to 6 s. The receiver gain was kept constant for all acquisitions. Proton spectra were referenced at the resonance peak of sodium maleate (5.95 ppm).
For the AF sample preparation, 400 μL D2O and 150 μL phosphate buffer in D2O were added to lyophilized AF samples. After centrifugation (4500× g, 15 °C, 5 min), 50 μL of sodium maleate was added as an internal standard to 500 μL of the supernatant, and the sample was transferred to 5 mm NMR tubes.
Sodium maleate was chosen as the reference standard since it is suitable for the CPMG pulse sequence and provides a distinct peak in the 1 H NMR spectrum. The samples were thawed at room temperature for 60 min before performing NMR experiments.
All NMR spectra were acquired using a Varian-600MHz NMR spectrometer equipped with a triple resonance probe {HCN} at 25 °C. The Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence was applied to AF samples with 128 transients collected with 64 K data points. The relaxation delay was set to 6 s. The receiver gain was kept constant for all acquisitions. Proton spectra were referenced at the resonance peak of sodium maleate (5.95 ppm).
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