All brain samples were prepared using a modified version of the protocol previously described by Graham et al. (2013) 26 and Bahado-Singh et al. (2016) 27 . In brief, all brain specimens were weighed and defrosted on ice. Subsequently, samples were milled and extracted in 50 % methanol/water (1 g/ml) in a sterile 2 ml Eppendorf tube. Samples were mixed for 20 min, sonicated for 15 min and the protein removed via centrifugation at 13,000 g at 4 ºC for 30 min. Supernatants were collected and dried under vacuum using a Savant DNA Speedvac (Thermo Scientific, USA) and reconstituted in 285 μL of 50 mM potassium phosphate buffer (pH 7.0), 30 μL of Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) and 35 μL of D2O 28 . 200 μL of the reconstituted sample was transferred to a 3 mm Bruker NMR tube for NMR analysis. All samples were housed at 4°C in a thermostatically controlled SampleJet autosampler (Bruker-Biospin, USA) and heated to room temperature over 3 min prior to analysis by NMR.
Savant dna speedvac
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Savant DNA Speedvac is a laboratory vacuum concentrator designed to gently and efficiently remove solvents from samples. It utilizes a controlled temperature and vacuum to safely concentrate samples while preserving their integrity.
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2 protocols using savant dna speedvac
1
Brain Metabolite Extraction and NMR Analysis
2
Metabolite Extraction for 1H-NMR Analysis
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For 1H-NMR, 50 mg samples were extracted in 50% methanol/water (1 g/mL) in a sterile 2 mL Eppendorf tube. The samples were mixed for 20 min and sonicated for 20 min, and the protein was removed by centrifugation at 13,000× g at 4 °C for 30 min. Supernatants were collected, dried under vacuum using a Savant DNA Speedvac (Thermo Scientific, Waltham, MA, USA), and reconstituted in 285 μL of 50 mM potassium phosphate buffer (pH 7.0), 30 μL of Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) and 35 μL of D2O. Then, 200 μL of the reconstituted sample was transferred to a 3 mm Bruker NMR tube for analysis. All samples were housed at 4 °C in a thermostatically controlled SampleJet autosampler (Bruker-Biospin, Billerica, MA, USA) and heated to room temperature over 3 min prior to analysis by NMR [65 (link)].
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