A protocol utilising cold methanol water was used for the extraction of cytoplasmic metabolites from harvested bacterial cells (Maharjan and Ferenci, 2003 (link)). The previously lyophilised and weighed cell mass (c. 10 mg) was suspended in 10 ml of 50% cold methanol water, vortexed and then frozen in liquid nitrogen. It was allowed to thaw in ice for 10 minutes and the contents centrifuged to remove cell debris. The supernatant was removed and placed in a clean 50 ml Falcon tube. The methanol extraction was repeated on the pellet residue and centrifuged before combining the second supernatant with the first supernatant. Tubes containing the supernatant extract were dried in a Labconco centrifugal vacuum concentrator to remove the methanol/water. The residue was derivatised appropriately for analysis by gas chromatography with flame ionisation detection (GC-FID).
Centrifugal vacuum concentrator
Manufactured by Labconco
Sourced in United States
The Centrifugal Vacuum Concentrator is a laboratory instrument designed to concentrate samples by removing solvents or other volatile components through the application of centrifugal force and vacuum. It provides a controlled environment for the evaporation process, preserving the integrity of the sample.
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6 protocols using centrifugal vacuum concentrator
1
Extraction of Bacterial Metabolites
2
Extraction and Analysis of Wheat Compounds
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Samples were extracted according to Zaupa et al. [39 (link)] with some modifications. For instance, 1.5 mL of ethanol/water/formic acid (97:2:1, v/v/v) mixture was added to 150 mg of ground wheat. The samples were extracted for 15 min using a platform shaker (Ika Werke, Breisgau, Germany) at a speed of 200 strokes/min and subsequently centrifugated for 10 min at 10,000 rpm (radius 9.5 cm) at 4 °C. Next, 1 mL of clear supernatant was evaporated to dryness using a centrifugal vacuum concentrator (Labconco, Kansas City, MO, USA) and re-dissolved in a mixture of 100 µL of methanol/water (50:50, v/v) and then transferred into vials prior to being injected into the UHPLC-MS/MS system.
3
Mass Spectrometry Proteomics of Extracellular Vesicles
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After solubilized in lysis buffer (8 M urea, 50 mM NH4HCO3), EVs proteins were reduced with 20 mM dithiothreitol for 1 h at 37°C and alkylated with 40 mM iodoacetamide for 45 min at room temperature in the dark. For protein digestion, Lys C was first added at an enzyme/protein ratio of 1:50 (w/w) for 3 h at 37°C. After diluting with 25 mM NH4HCO3 to the final concentration of <1.5 M urea, the samples were further digested at 37°C overnight with trypsin at an enzyme/protein ratio of 1:50 (w/w). The digestion was then stopped by adding formic acid to a final concentration of 0.1% and desalted with a C18 Oasis HLB cartridge (Waters, USA). All peptide samples were dried in a centrifugal vacuum concentrator (Labconco, USA).
4
HPLC-ELSD Analysis of Plant Sugars
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Sugar analysis was conducted with HPLC-ELSD according to a protocol described in O’Donoghue et al. [16 (link)], with minor modifications. Briefly, 20 mg of pulverized material was extracted in three independent replicates with 2 mL of 75% methanol (v/v) for 15 min. After centrifugation at 14,500× g for 10 min, 750 µL of the supernatant was completely evaporated using a centrifugal vacuum concentrator (Labconco, Kansas City, MO, USA) at 40 °C. Upon reconstitution in 750 µL of water and subsequent filtration through a 0.22 µm nylon syringe filter (Chromservis), 10 µL of the sample was injected on a WATREX polymer IEX Ca-form 8 × 300 mm, 8 µm particle size column (WATREX, Prague, Czech Republic). The Smartline HPLC system (Knauer, Berlin, Germany) was coupled with an Alltech 3300 ELSD detector (Thermo Fisher). Sucrose, glucose, and fructose were identified and quantified using authentic standards (Lach-Ner, Neratovice, Czech Republic). MilliQ water (Milipore Sigma, Billerica, MA, USA), at a flow rate of 0.5 mL min−1, was used as the mobile phase. The ELSD detector operated at a temperature of 80 °C and a nitrogen flow rate of 2 L h−1. The analysis had a total run time of 20 min.
5
Pharmacokinetics and Tissue Distribution of Remdesivir Metabolites
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Normal BALB/c mice (male, 18–22 g) were treated with Rdv-cyc (intravenous injection), Rdv-cyc (transtracheal injection), or Rdv-lips (transtracheal injection) at a remdesivir dosage of 20 mg/kg per mouse. For the determination of NTP pharmacokinetics in the lung, the mice (n = 3) were ordinally sacrificed to isolate the lung tissue at predetermined time points (1, 2, 4, 8, 12 and 24 h). Precold extraction buffer containing 0.1% potassium hydroxide and 67 mM EDTA in 70% methanol [14] (link) and 0.2 µM sofosbuvir triphosphate (internal standard) was added and homogenized. The homogenate was centrifuged at 20 000 g for 20 min, and then the supernatants were collected and dried in a centrifugal vacuum concentrator (Labconco Corporation, USA). The dried samples were reconstituted with 100 µl mobile phase and then centrifuged at 20 000 g for 20 min. The NTP concentration in supernatants was determined by LC/MS/MS experiments, which details were provided in the supplementary information. For the determination of NTP tissue distribution, the mice (n = 6) were ordinally sacrificed to isolate the tissue (the heart, liver, spleen, lung, kidney, brain, and testis) at predetermined time points (4 h and 24 h). The sample treatment method and LC/MS/MS method were the same as above.
6
Protein Extraction and Digestion
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The cell lysates or EV samples were precipitated with acetone and resolved in 8 M urea with 50 mM Tris‐HCl (pH = 8.0). The samples were reduced by 10 mM dithiothreitol, alkylated by 10 mM iodoacetamide (45 min at RT in the dark), and digested with sequencing‐grade modified trypsin (Promega) at a substrate ratio of 1:50 (w/w) overnight at 37 °C. Then, the peptide sample was desalted with a C18 Oasis HLB cartridge (Waters, USA) and dried in a centrifugal vacuum concentrator (Labconco, MI, USA).
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