Total AAs composition (Arginine, Serine, Valine, Methionine, Leucine, Alanine, Tyrosine, Threonine, Proline, Lysine, Isoleucine, Histidine, Phenylalanine, Glycine, Glutamate, Aspartate, and Cystine) were determined following an international standard EN ISO 13903, using GC-FID ion chromatography with the electrochemical detector, manufactured by Thermo, model ICS-5000, with a silver reference electrode (Ag/AgCl) and gold (Au) working electrode and chromatographic column AminoPac PA10 and pre-column AminoPac PA10 guard.
Aminopac pa10
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Aminopac PA10 is an ion exchange chromatography column designed for the analysis of amino acids. It provides high-resolution separation of amino acids in a variety of sample types. The column is constructed with a polymeric resin and is compatible with common amino acid analysis methods.
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6 protocols using aminopac pa10
1
Amino Acid Composition Analysis by GC-FID
2
Amino acid analysis of fish sauce
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An analysis of free amino acids was performed using the ICS6000 chromatographic system (Thermo Fisher Scientific S.p.A, Milano, Italy). Samples of fish sauce were diluted 1:2000 with ultra-pure water before the injection in the chromatographic apparatus. The separation was carried out at 30 °C with an Aminopac PA10 analytical column (250 × 2 mm, 8.5 μm particle size) (Thermo Fisher Scientific S.p.A, Milano, Italy), using NaOH 250 mM and sodium acetate 1 M as eluent water, and a time/potential waveform according to the conditions shown in Table 1 .
3
Hydrolysis and Amino Acid Analysis of Apple
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Apple wounds were inoculated as described for RT-qPCR, and samples for analysis were withdrawn at 24, 36, 48, 72, and 144 h of incubation. Samples were treated by acid hydrolysis as follows: 250 mg of lyophilized apple was hydrolyzed with 20 mL of 6 N HCl at 110 °C for 24 h. The hydrolyzed samples were filtered, washed with ultrapure water, evaporated and then re-dissolved in 0.1 N HCl. All samples were tenfold diluted with ultrapure water and analyzed by an ICS6000 chromatographic system (Thermo Fisher Scientific S.p.A, Milano, Italy). Separation was performed with an Aminopac PA10 analytical column (250 × 2 mm, 8.5 μm particle size) (Thermo Fisher Scientific S.p.A, Milano, Italy). The chromatographic separation of the amino acids was performed using as eluents ultra-pure water, 250 mM NaOH and 1 M NaOAC with a flow rate of 0.25 mL/min using conditions shown in Supplementary Table 3 .
4
Monosaccharide Composition Analysis via HPAEC-PAD
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Monosaccharide composition of saccharides was determined by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Thermo Fisher ICS-5000+, USA) according to the method proposed by Li et al. (2021) (link). Briefly, saccharides sample (10 mg) was submitted to hydrolysis with 2 mL of 2.0 mol/L trifluoroacetic acid (TFA) at 80 °C for 4 h. After removing the excess of TFA by rotary evaporation, sample volume was adjusted to 10 mL with deionized water. Subsequently, the samples was purified using SupelClean™ LC-18 tubes (500 mg/6 mL) (Supelco, USA) and 0.22 μm filters, and then analyzed in an AminoPac™ PA10 (Dionex, 3 × 250 mm) column with 17 mmol/L NaOH at 30 °C at a flow rate of 0.25 mL/min.
5
Determination of γ-Carboxyglutamic Acid
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γ-Carboxyglutamic acid (Gla) content was determined by amino acid analysis as described by Price [19 (link)]. One mg of protein was subjected to alkaline hydrolysis in 2 N KOH for 48 h at 110°C. Hydrolyzed amino acids were filtrated through a 0.2 μm filter to remove precipitate. Supernatants were separated by high performance liquid chromatography on an AminoPac PA10 strong anion-exchange column with pulsed amperometric detector (Dionex, Sunnyvale, CA) using an elution buffer of 1 M sodium citrate. Purified γ-carboxyglutamic acid and aspartic acid (Sigma-Aldrich) were used as standards, and samples were compared with a control sample (pdFIX, Enzyme Research Labs, South Bend, IN). Determinations were performed in duplicate, and results are reported as an average.
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γ-Carboxyglutamic acid (Gla) content was determined by amino acid analysis as described by Price [19 (link)]. One mg of protein was subjected to alkaline hydrolysis in 2 N KOH for 48 h at 110°C. Hydrolyzed amino acids were filtrated through a 0.2 μm filter to remove precipitate. Supernatants were separated by high performance liquid chromatography on an AminoPac PA10 strong anion-exchange column with pulsed amperometric detector (Dionex, Sunnyvale, CA) using an elution buffer of 1 M sodium citrate. Purified γ-carboxyglutamic acid and aspartic acid (Sigma-Aldrich) were used as standards, and samples were compared with a control sample (pdFIX, Enzyme Research Labs, South Bend, IN). Determinations were performed in duplicate, and results are reported as an average.
6
Automated Synthesis of 18F-Fluciclovine PET Tracer
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18F-Fluciclovine was produced on a FASTlab synthesiser, using single use cassette kits provided by Blue Earth Diagnostics Limited. The 18F-Fluciclovine (or 18F- FACBC) was produced with a radioactivity yield of 49.1 ± 3.8% (n = 4) and the end of synthesis activity concentration of 1481 ± 190 MBq/ml. At end of synthesis the radiochemical purity was found to be 99.18 ± 0.13% (n = 4, Additional file 1 : Figure S1), and the molar activity was found to be 1044.45 ± 638.14 GBq/µmol (n = 3). The final 18F-Fluciclovine product was formulated in citrate buffer and was terminally filtered through a 0.2-µm filter.
Radiochemical purity was measured using thin-layer chromatography. Thin-layer chromatography silica gel 60 aluminium sheets strips were eluted over 7.5 cm using a mobile phase consisting of acetonitrile:methanol:water:acetic acid at 20:5:5:1 v/v.
Fluciclovine content was measured using ion chromatography and electrochemical detection with an amino acid waveform, a gold working electrode and a Ag/AgCl reference electrode. Analysis was performed on a Dionex AminoPac PA10 analytical column (4 × 250 mm) eluted with 150 mM NaOH.
Radiochemical purity was measured using thin-layer chromatography. Thin-layer chromatography silica gel 60 aluminium sheets strips were eluted over 7.5 cm using a mobile phase consisting of acetonitrile:methanol:water:acetic acid at 20:5:5:1 v/v.
Fluciclovine content was measured using ion chromatography and electrochemical detection with an amino acid waveform, a gold working electrode and a Ag/AgCl reference electrode. Analysis was performed on a Dionex AminoPac PA10 analytical column (4 × 250 mm) eluted with 150 mM NaOH.
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