20 μM tRNA was digested overnight in 100 μl of 25 mM HEPES pH 7.5, 200 mM NaCl, 0.1 mM ZnSO4 at 37°C by nuclease P1 (2 units, Sigma) followed by the addition of alkaline phosphatase for 2h at 37°C (2 units, Sigma). HPLC-tandem mass spectrometry analyses were performed with an ExionLC chromatographic system coupled with a QTRAP6500 + mass spectrometer (AB SCIEX INSTRUMENTS) equipped with a Turbo Spray IonDrive source used in the positive ionization mode. HPLC separation was carried out with a 2 × 150 mm, 2.7 μm Poroshell HPH-C18 column (Agilent, France) at 0.4 ml.min−1 and 35°C. A linear gradient of 0–15% acetonitrile in 0.1% formic acid over 7 min was used as the mobile phase. Mass spectrometry detection was carried out in the multiple reactions monitoring mode to obtain high sensitivity and specificity. The transitions used to quantify s4U were 261→129 and 261→112, corresponding to the loss of ribose. Under the HPLC conditions used, s2U and s4U are well separated, with s2U eluting faster (4.3 min) than s4U (4.6 min). Quantification was performed by external calibration.
Poroshell hph c18 column
Manufactured by Agilent Technologies
Sourced in United States
The Poroshell HPH-C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of analytes. It features a superficially porous particle technology that provides high efficiency and fast separations. The column is suitable for a variety of applications, including pharmaceutical, environmental, and food analysis.
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Lab products found in correlation
Nuclease p1, by Merck Group (1 mentions)
Qtrap 6500 mass spectrometer, by AB Sciex (1 mentions)
Alkaline phosphatase, by Merck Group (1 mentions)
Hp chemstation software, by Agilent Technologies (1 mentions)
Qtrap 5500 mass spectrometer, by AB Sciex (1 mentions)
Analyst software, by AB Sciex (1 mentions)
Lc 20, by Shimadzu (1 mentions)
Protein a biosensor, by Molecular Devices (1 mentions)
Ysi 2900d, by Yellow Springs Instruments (1 mentions)
Ak00091, by NZYTech (1 mentions)
Octet red, by Molecular Devices (1 mentions)
1260 hplc, by Agilent Technologies (1 mentions)
Agilent 1290 infinity uhplc system, by Agilent Technologies (1 mentions)
Agilent 1100, by Agilent Technologies (1 mentions)
Iodoacetic acid, by Merck Group (1 mentions)
O phthaldialdehyde opa, by Merck Group (1 mentions)
Norvaline, by Merck Group (1 mentions)
Sarcosine, by Merck Group (1 mentions)
Fluorenylmethyloxycarbonyl chloride, by Merck Group (1 mentions)
Agilent 1260 system, by Agilent Technologies (1 mentions)
12 protocols using poroshell hph c18 column
1
Quantification of tRNA Modifications
2
BLA GABA and Glutamate Quantification
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Tissue concentrations of GLU and GABA were measured by high-performance liquid chromatography (HPLC) with a fluorescence detector (Chen et al., 2019 (link)). Samples of the BLA (10 mg) were homogenized in saline solution (1 g: 9 ml). Twenty microliter of BLA homogenate was mixed with 70 μl 0.4 M perchloric acid, and then centrifuged at 12,000 g and 4°C for 20 min. The supernatants were removed for analysis and filtered through a 0.22 mm GV filter (Millipore, Bedford, MA, USA). Then, 10 μl of each sample was injected into an Agilent Poroshell HPH-C18 column (4.6 mm × 50 mm, 2.7 μm) through a temperature-controlled autosampler (Agilent). The mobile phase was composed of 10 mM disodium hydrogen phosphate buffer and 10 mM sodium borate buffer in water, methanol and acetonitrile (78:13:9, v/v). The flow rate was kept constant at 1 ml·min−1. Chromatographic analyses were performed at the column temperature of 35°C. Fluorescence detector as operated using an excitation wavelength of 355 nm and an emission wavelength of 450 nm. Methanol was used as an internal standard, and the neurochemicals of interest were quantified using HP ChemStation software (Agilent, St. Clara, CA, USA). The excitation/inhibition (E/I) ratio was defined as the GLU/GABA ratio.
3
Quantification of Mycotoxins by UHPLC-MS/MS
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UHPLC-ESI-MS/MS analysis was performed as described previously [31 (link)]. The UHPLC system was operated using Shimadzu LC20 (Kyoto, Japan). C18 column (2.1 × 50 mm, 1.9 μm) and C18 guard column (inner diameter 2.1 × 5 mm, 1.9 μm) were used to separate ZEN, Z14G, α-ZEL, and β-ZEL on an Agilent Poroshell HPH-C18 column at 40 °C. The elution gradient was applied as follows: 0 min, 80% B; 2 min, 80% B; 10 min, 30% B; 12 min, 30% B; 14 min, 80% B; 16 min, 80% B. The mobile phase consists of (A) acetonitrile containing 0.1% formic acid and (B) double-distilled water containing 0.1% formic acid, with a flow rate of 0.3 mL/min. The injection volume was 5 μL. The mass system was performed using a QTRAP 5500 mass spectrometer (AB SCIEX, Framingham, MA, USA). MS/MS detection was operated in both positive and negative ESI modes. The equipment parameters were as follows: ion spray temperature (TEM): 450 °C; ion spray voltage (IS): −4500 V (in MRM−mode)/5500 V (in MRM+ mode); ion source gas1 (GS1): 55 psi; ion source gas2 (GS2): 55 psi; curtain gas (CUR): 35 psi; collisional activated dissociation (CAD) gas: medium level. The detection parameters of ZEN, Z14G, α-ZEL, and β-ZEL are shown in Table S1 . Analyst software (AB Sciex, Redwood city, CA, USA., version 1.6.0) was used for data analysis.
4
Comprehensive Bioanalytical Characterization
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Concentrations of glucose (c Glc c Gln c Lac c Amm c Ab 0.5 μ m 38 ].
5
HPLC Analysis of ILQ Compound
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ILQ was assayed by reversed phase HPLC on a Poroshell HPH-C18 column (250 × 4.6 mm, 4 μm, Agilent Co. Ltd., USA). The HPLC was performed on a waters e2695 system consisting of alliance Quat gradient pump, 2998 diode array detector and empower 3 chromatographic software. The HPLC method for analyzing ILQ was established as previously reported.32 (link) The detailed conditions were as follows: acetonitrile-water mixture (60/40, v/v) was used as mobile phase, with column temperature at 30°C, wavelength at 372 nm, and flow rate 1.0 mL/min.
6
Methionine Oxidation in Irradiated Samples
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Three months after irradiation, bags were filled with a 50 µM solution of methionine in buffer (10 mM NaH2PO4, 10 mM Na2B4O7•10H2O, 5 mM NaN3, pH 8.2). After storage for 10 days sampling was performed and the solution analyzed with an Agilent 1260 HPLC equipped with a quaternary pump (G1311C), an autosampler (G1329B), and a fluorescence detector (G1321B). Separation between the methionine and its sulfoxide form was carried out on an Agilent Poroshell HPH-C18 column (4.6 mm × 100 mm, 2.7 μm particles) used with a UHPLC guard Poroshell HPH-C18, 4.6 mm pre-column. Details of chemicals and reagents used, HPLC system and conditions are described in a previous article (Girard-Perier et al., 2020a (link)).
7
Extensive Fractionation for Complex Proteome Analysis
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Extensive fractionation by basic reverse-phase liquid chromatography was used to reduce sample complexity and thus reduce the likelihood of peptides being co-isolated and co-fragmented. The 11plex TMT labeled sample was resuspended in 20 μL of 10 mM TEAB (triethylammonium bicarbonate) buffer before being separated on an Agilent 1290 Infinity UHPLC system mounted with a Poroshell HPH-C18 Column (2.1 × 150 mm, 2.7 μm, Agilent). The labeled peptides were eluted using a gradient of 2–100% Solvent B (10 mM TEAB, pH 8.0, 90% ACN) at a flow rate of 200 μL/min over 120 min. A total of 96 fractions were collected in a 96 well plate, which were then concatenated to 24 fractions by combining every four sequential elutes, vacuum dried and stored at −80°C until further LC/MS-MS analysis.
8
Comprehensive Proteomics Profiling of HeLa Cells
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Protein lysates of wild-type HeLa cells treated with 100μMGA (or DMSO control) for 48 h and those with SHMT2 knockout (treated with NS) were prepared in triplicate. Each protein sample was diluted to 1 μg/μL (50 μg in all) with 100 mM tetraethylammonium bromide, reduced with 200 mM Tris(2-carboxyethyl)phosphine), alkylated with 375 mM iodoacetamide, precipitated with methanol, chloroform and water (CH3OH:CHCl3:H2O = 4:1:3), and subjected to overnight digestion with trypsin (1:50 enzyme to protein ratio). The tryptic peptides were labeled with 9 plexes of TMT10 reagents according to the manufacturer’s protocol. Then, the TMT-labeled peptides were mixed and fractionated by an Agilent Poroshell HPH C18 column (250 × 4.6 mm, OD 4 μm) at a flow rate of 1 mL/min on an Agilent 1260 instrument. Buffer A (2% ACN, 10 mM NH4COOH, pH = 10) and a non-linear increasing concentration of buffer B (90% ACN, 10 mM NH4COOH, pH = 10) were used for peptide separation. A standard 120 min gradient was used as follows: 0–8% B in 10 min; 8%–35% B in 70 min; 35%–60% B in 15 min; 60%–70% B in 10 min; and 70–100% B in 15 min. The peptide mixture was separated into 120 fractions and combined by a concatenation strategy into 30 fractions (1&31, 2&32 … 30&120). The combined fractions were used for further LC-MS/MS analysis.
9
Amino Acid Quantification via HPLC
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Quantification of individual amino acids was performed by high performance liquid chromatography (HPLC), Agilent 1100 (Agilent Technologies, Waldbronn, Germany) using pre-column derivatization and fluorescence detection based upon a method previously described (Henderson and Brooks, 2010 ) with some modifications to the derivatization and injection. A Poroshell HPH-C18 column (4.6 × 150 mm, 2.7 μm particle size; Agilent Technologies) was used following derivatization of the amino acids. Derivatization was performed using three different reagents: iodoacetic acid (Sigma Aldrich) for cysteine, o-phthaldialdehyde (OPA, Sigma Aldrich) for primary amino acids, and fluorenylmethyloxycarbonyl chloride (Sigma Aldrich) for secondary amino acids. Internal standards, norvaline (Sigma Aldrich), and sarcosine (Sigma Aldrich) were spiked to each sample prior to derivatization. One milliliter of each filtered sample was analyzed.
10
Agilent HPLC Fluorescence Separation
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Chromatography was performed using the Agilent 1260 system equipped with a degasser (Agilent Technologies Santa Clara, CA) (model G1379), a quaternary pump (model G1311X), a temperature-controlled autosampler (model G1329B), column oven enabling temperature control of the analytical column (model G1316A) and a fluorescence detection (model G1321A). All chromatographic separations were performed on an Agilent Poroshell HPH-C18 column (4.6 mm × 50 mm, 2.7 μm).
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