Culture broth was sampled rapidly and filtered through a 0.5-μm pore size filter (PTFE-type membrane, ADVANTEC, Japan). Cells on the filter were immediately immersed in 1.6 mL methanol (-80°C) and kept at -80°C until extraction. Following addition of 1.6 mL of chloroform (-30°C) and 640 μL of Milli-Q water (4°C) and vortexing for 1 min, the mixture was centrifuged at 15,000 g for 5 min at 4°C. Two hundred and fifty microliters of the aqueous layer was transferred to a 1.5-mL tube and dried using a SpeedVac SPD1010 (Thermo Scientific, Japan) at room temperature. The dried samples were suspended in 50 μL of Milli-Q water. LC-MS/MS analysis (LC: Agilent 1100 series; Agilent Technologies, MS/MS: API 2000; MA, AB SCIEX) was performed using the previously described method (17). The peak of each target metabolite was identified by comparing its chromatographic behavior with that of an authentic standard. Peak area was determined using the software Analyst (version 1.6.2, AB SCIEX).
Speedvac spd1010
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SpeedVac SPD1010 is a benchtop centrifugal evaporator designed for the rapid removal of solvents and water from samples. It uses a high-vacuum system to gently concentrate samples, preserving sample integrity. The unit is equipped with a temperature-controlled drying chamber and a refrigerated condensing system to efficiently trap and collect the evaporated solvents.
Automatically generated - may contain errors
Lab products found in correlation
Multi beads shocker, by Yasui Kikai (3 mentions)
Millex filter unit, by Merck Group (3 mentions)
Lc ms grade water, by Fujifilm (3 mentions)
Agilent 1100 series, by Agilent Technologies (1 mentions)
1290 hplc system, by Agilent Technologies (1 mentions)
6460 triple quad mass spectrometer, by Agilent Technologies (1 mentions)
Mass hunter quantitative software, by Agilent Technologies (1 mentions)
Omnipore filter disks, by Merck Group (1 mentions)
Lcms 8030 plus, by Shimadzu (1 mentions)
Nexera uplc system, by Shimadzu (1 mentions)
6 protocols using speedvac spd1010
1
Metabolite Extraction and Analysis
2
Quantification of Quinolinic Acid in Yeast
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For metabolite extraction, yeast cells (1 × 108 cells) were suspended in 50% methanol and immediately frozen in liquid nitrogen. Then, frozen samples were ground by a Multi Beads Shocker (Yasui Kikai, Osaka, Japan) then centrifuged at 13,000 × g for 10 min at 4 °C. The supernatant was mixed with an equal volume of chloroform, and the mixture was centrifuged again. The upper aqueous phase was transferred to a tube and evaporated using SpeedVac SPD 1010 (Thermo Fischer Scientific, Waltham, MA, USA). Levels of quinolinic acid (QUIN) in yeast cells were determined using an Agilent 6460 Triple Quad mass spectrometer coupled to an Agilent 1290 HPLC system with multiple reaction monitoring (MRM) mode. The MRM transition for QA was optimized as m/z 166 to 78. MS settings and chromatographic conditions were described previously73 (link). The amount of QA was calculated by integrating the sum of the area using Mass Hunter Quantitative software (Agilent Technologies, Santa Clara, CA, USA).
3
Metabolite Extraction and Derivatization
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Whole tissues were ground using Multibeads shocker (Yasui Kikai) with LC/MS‐grade methanol and water in equal proportion by volume and then centrifuges at 13,000 g for 5 min at 4°C. Supernatant was mixed with an equal volume of chloroform. The upper aqueous phase was dried using Speedvac SPD 1010 (Thermo). For LC/MS experiments, the dried pellet of sample was dissolved in 60 μl LC/MS‐grade water (Wako) and then passed through a filter with 0.45‐μm Millex filter unit (Millipore). For GC/MS experiments, the derivatization of samples was carried out in two steps. In the first step, carbonyl functional groups were protected by methoximation using 20 μl of 20 mg/ml solution of methoxyamine hydrochloride in pyridine at 30°C for 90 min. In the second step, after adding 80 μl N‐methyl‐N‐trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS; Pierce), samples were incubated at 37°C for 30 min for derivatization.
4
Comprehensive metabolite profiling by LC-MS and GC-MS
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Whole tissues were grinded by Multi Beads Shocker (Yasui Kikai) with LC-MS grade methanol and water in the proportion of 1:1 (by volume). After the centrifugation, the supernatant was mixed with the same volume of chloroform, and the aqueous phase was dried by SpeedVac SPD1010 (Thermo). For the LC/MS analysis, the dried sample was reconstituted by 50 μl LC/MS grade water (Wako) and filtered with 0.45 μm Millex filter unit (Millipore). For the GC/MS analysis, two-step derivatization was carried out. First, carbonyl functional groups were protected by methoximation using
20 μL of 20 mg/mL solution of methoxyamine hydrochloride in pyridine at 30°C for 90 min. Next, the samples were derivatized using 80 μL of N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS, Pierce) at 37°C for 30 min.
5
13C-labeling Kinetics in Metabolite Profiling
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After NaH 13 CO3 was added, 10 mL of culture was filtered at each time point (0, 1, 2, 3, 4, 5, 10, 15, 30, and 60 min) using 0.2 μm pore size Omnipore filter disks (Merck KGaA).
The harvested cells on the filter were immediately immersed in pre-chilled methanol containing 5 μM (+)-10-camphorsulfonate to quench metabolic activity. Quenching was performed for less than 15 sec. The mixture was stored at -80°C until extraction.
Intracellular metabolites in the mixture were extracted using chloroform and Milli-Q water. The supernatant containing intracellular metabolites was collected and concentrated using a centrifugal concentrator SpeedVac SPD1010 (Thermo Fisher Scientific, Waltham, MA, USA). The dried samples were resuspended in 100 μL Milli-Q water for LC-MS/MS analysis. The resuspended solutions were used to measure 13 Clabeling patterns and pool sizes of metabolites using a Shimadzu Nexera UPLC system coupled with LCMS 8030 Plus (Shimadzu, Kyoto, Japan). The column used was a ProteCol-P C18 HQ103 (2.1 mm inside diameter × 150 mm, particle size of 3 μm; SGE Analytical Science, Melbourne, Australia). The chromatographic conditions were as described in a previous study (Dempo et al., 2014) . All data acquisition and analyses were performed using LabSolutions version 5.60 (Shimadzu).
The harvested cells on the filter were immediately immersed in pre-chilled methanol containing 5 μM (+)-10-camphorsulfonate to quench metabolic activity. Quenching was performed for less than 15 sec. The mixture was stored at -80°C until extraction.
Intracellular metabolites in the mixture were extracted using chloroform and Milli-Q water. The supernatant containing intracellular metabolites was collected and concentrated using a centrifugal concentrator SpeedVac SPD1010 (Thermo Fisher Scientific, Waltham, MA, USA). The dried samples were resuspended in 100 μL Milli-Q water for LC-MS/MS analysis. The resuspended solutions were used to measure 13 Clabeling patterns and pool sizes of metabolites using a Shimadzu Nexera UPLC system coupled with LCMS 8030 Plus (Shimadzu, Kyoto, Japan). The column used was a ProteCol-P C18 HQ103 (2.1 mm inside diameter × 150 mm, particle size of 3 μm; SGE Analytical Science, Melbourne, Australia). The chromatographic conditions were as described in a previous study (Dempo et al., 2014) . All data acquisition and analyses were performed using LabSolutions version 5.60 (Shimadzu).
6
Metabolite Extraction and Derivatization for LC-MS and GC-MS
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Metabolites were extracted from cells with water/methanol/chloroform (25:25:50 by volume). After centrifugation, the aqueous phase was isolated and dried using a SpeedVac SPD1010 (Thermo). For LC-MS analysis, the dried sample was reconstituted with 50 μl LC-MS grade water (Wako) and filtered through a 0.45 μm Millex filter unit (Millipore). For GC-MS analysis, two-step derivatization was carried out. First, carbonyl functional groups were protected by methoximation using 20 μL of 20 mg/mL methoxyamine hydrochloride in pyridine at 30°C for 90 min. Next, samples were derivatized using 90 μL of N-methyl-N-trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (MSTFA + 1% TMCS, Pierce) at 37°C for 30 min.
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