Two different extractions methods were performed using leaves of 4-week-old plants: (1) 10,000 tVI-GTs, hand-picked from leaves using a glass micropipette (Kang et al., 2010b (link)) under a dissection microscope, were extracted with 200 μL MeOH and (2) leaf disks (5 mm diameter) obtained with a hole puncher were immediately submerged in 1 mL MeOH. Extracts were analyzed on a Xevo TQD QQQ-MS (Waters, Broadway, NY, USA) following separation on an Ascentis Express C18 HPLC column (5 cm × 2.1 mm, 2.7 μm; Sigma-Aldrich, St Louis, MO, USA). Analytes were separated at a flow rate of 0.3 mL min−1 using a linear gradient from 65% solvent A (acetonitrile:water = 3:2, v:v) and 35% solvent B (acetonitrile:isopropanol = 1:9, v:v) to 100% solvent B at 4 min, followed by 100% B for another 1 min. Chlorophyll a and b were quantified by monitoring the m/z 893→ 555 and m/z 907 → 569 transitions, respectively. Bulk chlorophyll and carotenoid levels in leaf samples were determined spectrophotometrically as previously described (Lichtenthaler and Wellburn, 1983 ).
Ascentis express c18 hplc column
Manufactured by Merck Group
Sourced in United States, Japan
The Ascentis Express C18 HPLC column is a reversed-phase liquid chromatography column designed for high-performance separation. It features a solid-core particle technology that provides high efficiency and fast analysis times. The column is suitable for the separation and analysis of a wide range of compounds.
Automatically generated - may contain errors
Lab products found in correlation
Quanoptimize software, by Waters Corporation (4 mentions)
Prominence uflc xr system, by Shimadzu (3 mentions)
Xevo tq s tandem quadrupole mass spectrometer, by Waters Corporation (2 mentions)
Maxis 2 quadrupole time of flight mass spectrometer, by Bruker (2 mentions)
Acquity uplc, by Waters Corporation (2 mentions)
Xevo tq s triple quadrupole mass spectrometer, by Waters Corporation (2 mentions)
Securityguard c18 guard column, by Phenomenex (2 mentions)
Xcaliber quant browser, by Thermo Fisher Scientific (2 mentions)
Tsq quantum triple quadrupole mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Proteinpilot software 4, by AB Sciex (1 mentions)
Trypsin, by Promega (1 mentions)
Prominence hplc system, by Shimadzu (1 mentions)
Maxis 2, by Bruker (1 mentions)
12 protocols using ascentis express c18 hplc column
1
Quantitative Analysis of Chlorophyll and Carotenoids in Plant Leaves
2
Quantification of Oxylipids by LC-MS/MS
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In short, the quantification of metabolites was accomplished on a Waters Xevo-TQ-S tandem quadrupole mass spectrometer using multiple reaction monitoring. Chromatography separation was performed with an Ascentis Express C18 HPLC column (Sigma-Aldrich, St. Louis, MO), held at 50°C and autosampler held at 10°C. Mobile phase bottle A was water containing 0.1% formic acid and mobile phase bottle B was acetonitrile; the flow rate was 0.3 mL/min. Liquid chromatography separation took 15 min per sample with linear gradient steps programmed as follows (A:3B ratio): time 0 to 0.5 min (99:1), to (60:40) at 2.0 min; to (20:80) at 8.0 min; to (1:99) at 9.0 min; 0.5 min held at (1:99) until min 13.0; then return to (99:1) at 13.01 min, and held at this condition until 15.0 min. All oxylipids were detected using electrospray ionization in negative-ion mode. Cone voltages and collision voltages were optimized for each analyte using Waters QuanOptimize software and data analysis was carried out with Waters MassLynx software.
3
Quantification of Isoprostanes via LC-MS/MS
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Details of LC/MS/MS analysis are described in Mavangira et al., 2016 [25 (link)]. In short, the quantification of metabolites was accomplished on a Waters Xevo-TQ-S tandem quadrupole mass spectrometer using multiple reaction monitoring (MRM). Chromatography separation was performed with an Ascentis Express C18 HPLC column (10 cm × 2.1 mm; 2.7 μm particles, Sigma-Aldrich, St. Louis, MO, USA) at 50 °C, with the autosampler at 10 °C. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile. Flow rate was fixed at 0.3 mL/ min. Liquid chromatography separation took 15 min per sample. MRM parameters including cone voltage, collision voltage, precursor ion, product ion, and dwell time were optimized based on Waters QuanOptimize software by flow injection of pure standard for each individual compound. Total IsoP concentrations were obtained by addition of IsoP detected in each sample type.
4
Collagen Peptide Sequence Characterization
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The CMA peptide sequence was confirmed by an N-terminal amino acid sequence analysis. The tryptic digest of the glyoxal-modified type III collagen was applied to the CMA affinity column as described above. The adsorbed peptide fraction was loaded onto an Ascentis Express C18 HPLC column (Supelco, Bellefonte, PA, USA), and the CMA peptide-containing fraction was collected. This sample was analyzed by a Procise 492 protein sequencer (Applied Biosystems, Invitrogen Co., Carlsbad, CA, USA) in the pulsed liquid mode.
5
Quantifying Prolyl 3-Hydroxylation in Zebrafish Collagen
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The purified collagen samples were digested with trypsin as described above to evaluate the relative abundance of prolyl 3-hydroxylation at specific sites in zebrafish type I collagen by LC-MS. The tryptic digests were analyzed by LC-MS on the maXis II quadrupole time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to the Shimadzu Prominence UFLC-XR system (Shimadzu, Kyoto, Japan) using the Ascentis Express C18 HPLC column (5 μm particle size, L × I.D. 150 mm × 2.1 mm; Supelco, Bellefonte, PA, USA). Site occupancy of prolyl 3-hydroxylation at respective modification sites was semi-quantitatively estimated by the relative peak area ratio of monoisotopic extracted ion chromatograms for each 3-Hyp variant of tryptic peptides containing the modification sites described previously [58 (link)].
6
Mass Spectrometry Analysis of Collagen
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Protein identification was performed using in-gel digestion as described previously [55 (link)]. The relative expression of collagen chains purified from the cortical bones from wild-type and Hand1-overexpressing mice was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 5% gel under non-reducing conditions. After staining with Coomassie Brilliant Blue R-250, the SDS-PAGE gel was scanned and the band intensity was measured by densitometric analysis using Multi Gauge version 3.0 (Fujifim, Tokyo, Japan). Protein bands of purified collagen samples were excised and digested in-gel with trypsin (Promega, Madison, WI, USA) at 37 °C for 16 h. The tryptic digests were analyzed by liquid chromatography–mass spectrometry on a maXis II quadrupole time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to a Shimadzu Prominence UFLC-XR system (Shimadzu, Kyoto, Japan) with chromatographic separation using an Ascentis Express C18 HPLC column (2.7 μm particle size, L × I.D. 150 mm × 2.1 mm; Supelco, Bellefonte, PA, USA) as described previously [56 (link)]. A database search was performed against the UniProtKB/Swiss-Prot database (release 2018_05) for Mus musculus species (16970 protein entries) using ProteinPilot software 4.0 (AB Sciex, Foster City, CA, USA).
7
Separation and Identification of OxPC Species
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Separation of OxPC species was done as described previously [25 (link)]. Briefly, 30 μL of sample extracts, reconstituted in reverse phase solvent, were injected onto an Ascentis Express C18 HPLC column (15 cm × 2.1 mm, 2.7 μm; Supelco Analytical, Bellefonte, Pennsylvania, USA) using a Prominence HPLC system (Shimadzu Corporation, Canby, Oregon, USA). Elution was performed by linear gradient of solvent A (acetonitrile/water, 60:40 vol/vol) and solvent B (isopropanol/acetonitrile, 90:10, vol/vol) both solvents containing 10 mM ammonium formate and 0.1% formic acid. The time program used was as follows: initial solvent B at 32%, increased to 45% B until 4.00 min; 5.00 min 52% B; 8.00 min 58% B; 11.00 min 66% B; 14.00 min 70% B; 18.00 min 75% B; 21.00 min 97% B; 25.00 min 97% B; 25.10 min 32% B until the elution was stopped at 30.10 min. A flow rate of 0.26 ml/min was used for analysis. The temperature of the column oven and sample tray was maintained at 45 and 4°C, respectively.
8
Quantifying Collagen Post-Translational Modifications
9
Quantitative Metabolite Analysis via UPLC-MS/MS
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The quantification of metabolites was accomplished on a Waters Acquity UPLC coupled to a Waters Xevo TQ-S triple quadrupole mass spectrometer (Waters) using multiple reaction monitoring. Chromatography separation was performed with an Ascentis Express C18 HPLC column, 10 cm × 2.1 mm, 2.7 μm (Supelco, Bellefonte, PA) held at 50°C. The autosampler was held at 10°C. Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile. Flow rate was fixed at 0.3 mL/min. Liquid chromatography separation took 15 min with linear gradient steps programmed as follows (A:B ratio): time 0 to 0.5 min (99:1), to (60:40) at 2.0 min; to (20:80) at 8.0 min; to (1:99) at 9.0 min; 0.5 min held at (1:99) until min 13.0; then return to (99:1) at 13.01 min, and held at this condition until 15.0 min. All oxylipids and fatty acids were detected using electrospray ionization in negativeion mode, whereas the endocannabinoids were detected using positive-ion mode. Cone voltages and collision voltages were optimized for each analyte using Waters QuanOptimize software (see Appendix for multiple reaction monitoring parameters).
10
Quantification of 15-F2t-Isoprostane by LC-MS/MS
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Details of liquid chromatography-tandem MS procedures are described in Mavangira et al. (2015) . Briefly, the quantification of 15-F 2t -isoprostane was accomplished on a Waters Acquity UPLC coupled to a Waters Xevo TQ-S triple quadrupole mass spectrometer using multiple reaction monitoring. Chromatography separation was performed with an Ascentis Express C18 HPLC column (10 cm × 2.1 mm, 2.7 µm; Supelco, Bellefonte, PA) held at 50°C. The autosampler was held at 10°C. Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile. Flow rate was fixed at 0.3 mL/min. Liquid chromatography separation took 15 min with linear gradient steps programmed as follows (A/B ratio): time 0 to 0.5 min (99/1), to (60/40) at 2.0 min; to (20/80) at 8.0 min; to (1/99) at 9.0 min; 0.5 min held at (1/99) until min 13.0; then return to (99/1) at 13.01 min, and held at this condition until 15.0 min. 15-F t2 -isoprostane was detected using electrospray ionization in negative-ion mode. Cone voltages and collision voltages were optimized using Waters QuanOptimize software. All samples were processed, extracted, measured, and analyzed as a single batch, minimizing intra-assay variation; however, a specific intra-assay variation coefficient is not available. Because a single batch of samples was assayed, an inter-assay variation coefficient does not apply.
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