Ten microliter of reconstituted extract was transferred to an autosampler vial and was combined with 70 μL of Borate Buffer (Waters, 186003836) from Waters AccQ-Tag Derivatization Kit (target pH: 8–10) and vortexed. Twenty microliter of AccQ-Tag Derivatization Agent (Waters, 186003836) was added, vortexed and let stand for 1 min. Samples were derivatized (55°C, 10 min) and vortexed. Derivatized samples were quantified with a Waters Acquity UPLC, Xevo-μ Tandem Mass Spectrometer and an AccQ-Tag Ultra RP Column 130 Å, 1.7 μm, 2.1 mm, 100 mm column using multiple reaction monitoring (MRM) and internal standard calibration (72 (link), 73 (link)).
Borate buffer
Manufactured by Waters Corporation
Sourced in United States
Borate buffer is a chemical solution commonly used in laboratory settings to maintain a specific pH range. It is a versatile buffer system that can be used in a variety of analytical and biochemical applications. The core function of borate buffer is to provide a stable and controlled pH environment for various experiments and procedures.
Automatically generated - may contain errors
Lab products found in correlation
Accq tag derivatization kit, by Waters Corporation (1 mentions)
O benzylhydroxylamine, by Merck Group (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide, by Merck Group (1 mentions)
Accq tag ultrac18, by Waters Corporation (1 mentions)
Acquity uplc h class system, by Waters Corporation (1 mentions)
Accq tag ultra eluent a, by Waters Corporation (1 mentions)
Accq tag ultra eluent b, by Waters Corporation (1 mentions)
4 protocols using borate buffer
1
UPLC-MS/MS Amino Acid Quantification
2
Amino Acid and Acylcarnitine Profiling of Human Plasma
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Using frozen, fasting samples, a 100 μL aliquot of human plasma was spiked with a 10 μL mixture of amino acid or acylcarnitine internal standard mixture and treated with bleach (to inactivate HIV particles) to a final concentration of 10% bleach. Samples were treated with 800 μL of ice-cold methanol and centrifuged to pellet precipitated protein. Amino acids were derivatized by drying down 100 μL of methanolic extract and reconstituting the sample with 80 μL of borate buffer (Waters Corp., Milford, MA) and 20 μL of MassTrak AAA Reagent Powder dissolved in MassTrak AAA Reagent Diluent (Waters Corp., Milford, MA) in a 96-well plate. Acylcarnitines were derivatized by reconstituting the dried methanolic extract in 100 μL of 0.2 M O-benzylhydroxylamine (Sigma–Aldrich, St. Louis, MO) and 10 μL of 2 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma–Aldrich, St. Louis, MO).
3
Amino Acid Content Determination
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Amino acid content determination was performed according to Cohen [73 ] with some modifications. Three milligrams of freeze-dried HC were suspended in 6 M HCl and 1% v/v phenol at 150 °C for 1 h. Hydrolysed samples were dissolved in 2 mL of 0.5 M citrate buffer. Amino acid content was determined by high-performance liquid chromatography (HPLC) in a Hewlett Packard model GmbH (Winchester, UK) connected to a fluorescence detector (Ex. 250 Em. 395). The derivation reaction was carried out with 20 µL of the sample diluted in 60 µL buffer (borate buffer, Waters, Thermo Scientific, Pierce™, MA, USA) and 1 min stirring. Twenty microlitres of reagent AQC (Waters) were added with stirring for another 1 min, followed by the heating of the sample at 50 °C for 10 min. The amino acid separation was carried out in a Bluespher® column (100 × 2 mm ID) in reverse phase C18 octa-decyl dimethylsilane (Berlin, Germany). Conditions of work: mobile phase A: 50 mM sodium acetate, pH 5.75 and mobile phase B: 50 mM sodium acetate, pH 6/CAN 30:70 v/v, and 1 mL/min flow.
4
Amino Acid Estimation in Cell Culture
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The Waters Acquity UPLC H class system was used for the spent medium amino acid estimation from the cell culture supernatant. The chromatography column AccQ.Tag Ultra C18 (1.7um, 2.1x 100mm, Waters, USA) was used with a flow rate of 0.7mL/minute, column temperature 43°C, sample storage temperature 2–8°C and UV detection at 260nm. The eluent mobile phase A (100% Waters AccQ.Tag Ultra Eluent A, Waters, USA), mobile phase B (10% Waters AccQ.Tag Ultra Eluent B in Milli-Q water, Waters, USA) mobile Phase C (Milli-Q water). For spent medium analysis, the harvest sample was filtered through 0.22μm syringe filter. Then took 10μL of sample/ amino acid standard (Waters, USA), 70μL of Borate buffer and 20μL of Derivatization reagent (Waters, USA) were mixed and heated at 55°C for 15 minutes. 1μl each sample and standard were injected on the above mentioned column. The amino acid standard used ranged from 10 to 250 pM for the quantitation of unknown amino acids. The Tryptophan is not available with Waters kit, therefore, tryptophan amino acid standard was prepared separately and the concentration of 100 to 1000nM was used for the estimation of unknown Tryptophan present in the cell culture media.
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