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16 protocols using ammonium acetate

1

Quantitative Proteomics Analysis Protocol

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LC grade methanol, acetonitrile, ammonium acetate and formic acid were purchased from CNW Technologies Inc. (CAS no: 67-56-1, 75-05-08, 631-61-8, 64-18-6, purity ≥ 99.9%, Shanghai, China). 2-Chloro-l-phenylalanine was obtained from Shanghai Heng-bai Biotech. Co. Ltd. (CAS no: 103616-893, purity ≥ 98%, Shanghai, China). Deionized water was produced by Mili-Q system (Millipore, Bedford, MA, USA). Antibodies against the following proteins were obtained for western blot: CS (Abcam, #96600, UK), EPRS (Abcam, #31513, UK), GAPDH (Cell Signaling, #5176, USA), horseradish peroxidase-labeled goat anti-rabbit secondary antibody was purchased from ZSGB-BIO (Beijing, China).
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2

Quantitative Analysis of Bioactive Compounds

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LC-MS grade acetonitrile, methanol, ammonium acetate, and formic acid were purchased from CNW Technologies (Duesseldorf, Germany). 2-Chloro-L-phenylalanine was purchased from Shanghai HB Biotech Co., Ltd. (Shanghai, China). Deionized water was prepared on a Millipore Milli-Q water purification system (Billerica, MA, USA). Apigenin was obtained from China National Engineering Research Centre for Solid Preparation Manufacturing Technology (Beijing, China). Psoralen was purchased from China Food and Drug Control Institute (Beijing, China). Assay kits for aminotransferase (ALT) and aspartate aminotransferase (AST) were purchased from Nanjing Jiancheng Biological Engineering (Nanjing, China). RFH was purchased from Baining Pharmaceutical Co., Ltd. (Guangzhou, Guangdong).
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3

UHPLC-QTOF-MS for Metabolite Profiling

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Ultra (high) performance liquid chromatography (UHPLC) separation was carried out using an ExionLC Infinity series UHPLC System (AB Sciex), equipped with a UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm, Waters). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide ( CNW Technologies) in water (pH = 9.75) (A) and acetonitrile (B). The analysis was carried out with an elution gradient. The column temperature was 25℃. The autosampler temperature was 4 ℃, and the injection volume was 2 μL (pos) or 2 μL (neg), respectively.
TripleTOF 5600 mass spectrometry (AB Sciex) was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during LC/MS experiments. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, the most intensive 12 precursor ions with intensity above 100 were chosen for MS/MS at a collision energy (CE) of 30 eV. In the detection process, standard products and blank controls were strictly used for quality control, and data quality control was also carried out. The UHPLC-QTOF-MS analysis was performed at Biomarker Technologies Corporation, Beijing, China (https://international.biocloud.net/).
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4

Ergosterol Purification and Characterization

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The ergosterol has been purified from the edible mushroom L. mongolica and and identified by ESI-MS and NMR (Zaki and Bao, 2022 ). The chemicals reagents were used in LC-MS grade acetonitrile, formic acid, methanol and ammonium acetate were bought from CNW Technologies (Duesseldorf, Germany). Deionized-ddH2O was obtained from the Millipore Milli-Q water purification system (Billerica, MA, USA). Acetic acid and methanol were obtained from Tedia Co. (Ohio, USA). Meanwhile, the Beyotime Institute of Biotechnology (Haimen, China) provided the cell counting kit-8 (CCK8) assay.
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5

PFAS Quantification Protocol

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Forty-one authentic standards and 13 internal standards were used in this study, as summarized in table S4. The solvents and reagents used in this study are of high-performance LC grade, including methanol from Merck (Germany), water from Thermo Fisher Scientific (USA), ammonium acetate from CNW technologies GmbH (Duesseldorf, Germany), and trichloroacetyl chloride from J&K (China). Standards were used to test mass accuracy, isotope accuracy, the recovery of our sample pretreatment method, and the synthesis and confirmation of PFAS.
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6

BPA Quantification by LC-MS/MS

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The BPA standard was purchased from Aldrich-Sigma (purity >98%, St. Louis, MO, USA). BPA-13 C12 (Cambridge Isotope Laboratories Ins, USA), HPLC-grade methanol, ethyl acetate and ammonium acetate (CNW Technologies) were purchased from Shanghai ANPEL Scientific Instrument Co., Ltd. β-Glucuronidase from Helix pomatia (Type HP-2, aqueous solution, β-glucuronidase activity ≥100000 units/mL, sulfatase activity ≤7500 units/mL) was purchased from Sigma-Aldrich. Deionized water for the preparation of buffers was purified by a Milli-Q ® water-purification system (Millipore, Bedford, MA, USA). An Oasis HLB cartridge (Waters, 200 mg/6 cm 3 , Milford, MA, USA) was purchased from Waters.
Stock solutions of BPA and 13 C12-BPA were prepared at a concentration of 1 mg/mL in methanol. All stock solutions were stored at -20°C. Calibration standards, ranging from 0.20 to 25 μg/L, were prepared from the stock solution through serial dilution by using a methanol-water mixture (50/50, v/v); isotope stock solutions ( 13 C12-BPA, 100 μg/L) were also prepared by using the same methanol-water mixture.
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7

UHPLC-MS/MS Analysis of Metabolites

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The UHPLC separation was conducted using a 1,290 Infinity series UHPLC System (Agilent Technologies, MA, United States) coupled to a UPLC BEH amide column (1.7 µm, 2.1 × 100 mm; Waters, Milford, United States). The mobile phase was composed of ammonium acetate (25 mmol/L, CNW Technologies, Shanghai, China) and ammonia hydroxide (25 mmol/L, CNW Technologies), which were added to water (pH = 9.75) (A) and acetonitrile (B), respectively. The following elution gradient was used for analysis: 0–0.5 min, 95% B; 0.5–7.0 min, 95%–65% B; 7.0–8.0 min, 65%–40% B; 8.0–9.0 min, 40% B; 9.0–9.1 min, 40%–95% B, and 9.1–12.0 min, 95% B. The injection volume, auto-sampler temperature, and column temperature were 2 µl (both positive and negative modes), 4°C, and 25°C, respectively (19 (link)).
MS/MS data acquisition and analysis were conducted using the Analyst TF v1.7 (AB Sciex, MA, United States) according to a preset standard. In each cycle, a collision energy (CE) of 30 eV and cycle time of 0.56 s was used to select the densest 12 precursor ions with an intensity >100 for MS/MS. The following were set as the electrospray ionization (ESI) source conditions: curtain gas (35 psi), gas 1 (60 psi), gas 2 (60 psi), ion spray voltage floating (5,000 V in the positive mode and −4,000 V in the negative mode), declustering potential (60 V), and source temperature (600°C) (19 (link)).
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8

Quantification of Oxidative Stress Markers

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Chemical carbon tetrachloride (CCl4) was purchased from Tianjin Tianli Chemical Reagent Co., Ltd. (Tianjin, China). Silymarin was obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). The diagnostic kits specific for necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were obtained from the Jiancheng Institute of Biotechnology (Nanjing, China). Chromatography-grade methanol, acetonitrile, ammonium hydroxide, and ammonium acetate were provided by CNW Technologies (Shanghai, China). All of the other chemicals and reagents used in the experiments were of analytical grade.
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9

Metabolomic Analysis of Botanical Samples

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Reagents including methanol, acetonitrile, ammonium acetate, ammonium hydroxide, and formic acid were purchased from CNW Technologies (ANPEL Laboratory Technologies, Inc. Shanghai, China). The internal standards of 2-chloro-L-phenylalanine was purchased from Shanghai Hengbai Biotech Co., Ltd. (Shanghai, China), and azadirachtin (>90%) were provided by Associate Professor Yongqing Tian at the South China Agricultural University.
Major instruments used in this study included ultra-high performance liquid chromatography (UHPLC) (Agilent Technologies, Santa Clara, CA, USA) coupled with Q Exactive Focus mass spectrometer (MS) (Thermo Fisher Scientific, Waltham, MA, USA), centrifuge, scales, grinding mill, water purification, ultrasound instrument, and column (ACQUITY UPLC HSS T3 1.7 μm, 2.1 × 100 mm).
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10

Dexamethasone Injection Quantification

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HPLC grade methanol, acetonitrile, ammonium acetate, and ammonium hydroxide were purchased from CNW Technologies (CNW, Germany). All aqueous solutions were prepared with ultrapure water produced by a Milli-Q water purification system (Millipore Corp., Billerica, MA, USA). Dexamethasone solution for injection was purchased from Shanghai Xinyi Pharmaceutical Co (Shanghai, China).
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