The amino acid levels in culture supernatants were analyzed by high-performance liquid chromatography (HPLC) using a previously described protocol [24 (link)] in which the proteins in the culture supernatant are first precipitated with methanol, and the amino acids are derivertized using o-phthalaldehyde (OPA) in an alkaline medium. Briefly, 200 μl of culture supernatants are added to an 800-μl mixture of methanol and internal standard (homocysteic acid). After centrifugation, the samples are loaded into the HPLC autosampler, which converts the samples to fluorescent derivatives (by mixing them with OPA) before their injection into the columns (C-18). A JASCO FP 1520 fluorescence detector at an excitation wavelength of 360 nm with emission detection at 455 nm was used to separate, detect, and quantify the fluorescent derivatives. For quantification of the intracellular amino acids, trophozoites (107) were lysed in 1 ml of trichloroacetic acid (TCA) 10% for 30 minutes at 4°C, and centrifuged, and the pH of the supernatants was adjusted to 12 using 10N NaOH. The amino acid concentration in the supernatants was then measured by HPLC on two biological replicates.
Fp 1520
Manufactured by Jasco
Sourced in Japan, United States, Germany, Canada
The FP-1520 is a laboratory instrument designed for fluorescence polarization measurements. It provides accurate and reliable data on the interactions between molecules, such as protein-ligand binding, enzyme-substrate interactions, and antibody-antigen complexes. The FP-1520 is a versatile tool for researchers in the fields of biochemistry, pharmacology, and drug discovery.
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Lab products found in correlation
Cba bond elute, by Agilent Technologies (3 mentions)
Waters chromatography system m600e, by Waters Corporation (2 mentions)
Eclipse xdb c18 column, by Agilent Technologies (2 mentions)
Spd 10av, by Shimadzu (2 mentions)
Azura hplc system, by Knauer (2 mentions)
Discovery c18 analytical column, by Merck Group (2 mentions)
Inertsil ods 4, by GL Sciences (1 mentions)
Pu 1580, by Jasco (1 mentions)
Ascentis c18 column, by Merck Group (1 mentions)
Prostar 410, by Shimadzu (1 mentions)
Alliance2695, by Jasco (1 mentions)
Atlantis t3 c18 reverse phase column, by Waters Corporation (1 mentions)
Lichrospher c18 guard column, by Merck Group (1 mentions)
Chromnav 2.0 hplc software, by Jasco (1 mentions)
Pu 4180 rhplc pump, by Jasco (1 mentions)
Prostar 410, by Jasco (1 mentions)
Zorbax eclipse, by Agilent Technologies (1 mentions)
Prism r, by Labnet (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Md 2018 plus, by Jasco (1 mentions)
28 protocols using fp 1520
1
Amino Acid Quantification by HPLC
2
Quantification of Metabolites in CAF Supernatants
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To determine the quantities of specific metabolites in supernatants of CAF, HPLC analyses were also carried out. Supernatants of CAF cultures were mixed with the internal standard 2‐oxovalerate and reacted with 10 mmol/L o‐phenylenediamine in 2‐mol/L HCl for 20 minutes at 80°C. Samples were desiccated with sodium sulfate, dried in vacuum, and re‐solubilized in methanol for HPLC analysis. Fluorescent derivatives were separated on an HPLC system comprising a pump (PU‐1580; JASCO, Tokyo, Japan), an auto sampler (AS920; JASCO), a column oven (CO980; JASCO), and a fluorescence detector (FP1520; JASCO). HPLC was carried out on an octadecylsilica column (Inertsil ODS‐4, 250 × 3.0 mm internal diameter, 5 μm; GL Sciences, Tokyo, Japan) at 40°C with a flow rate of 0.6 mL/min, using a mobile phase comprising water/methanol (55/45, v/v).
3
Quantification of Collagen Cross-Links
4
HPLC Quantification of L-Arginine
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The intracellular concentration of L-arginine was determined by HPLC using pre-column derivatization with o-phthalaldehyde (OPA) according to a previously published method,16 (link) with modifications. L-arginine (100 µM) was added to cell lysate (0.1 mM) as an internal standard. The samples were extracted on solid-phase extraction cartridges (CBA Bond Elute, Varian, Palo Alto, CA, USA). The recovery rate was 87.5±3.9%. Eluates were dried over nitrogen and resuspended in double-distilled water for HPLC analysis. HPLC was performed on a computer-controlled Waters chromatography system (M600E) consisting of an automatic injector (M7725i; Waters Co., Milford, MA, USA) and a fluorescence detector (FP-1520; Jasco Co., Portland, OR, USA). Samples were incubated for exactly 1 min with OPA reagent (5.4 mg/mL OPA in borate buffer, pH 8.4, containing 0.4% 2-mercaptoethanol) before automatic injection into the HPLC. The OPA derivatives of L-arginine and polyamine were separated on a 150×4.6 mm-5 µm Zorbax Eclipse XDB-C18 column (Watertown, MA, USA), with the fluorescence detector set at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. Samples were eluted from the column with 0.96% citric acid/methanol (70:30; pH 6.8) at a flow rate of 1.5 mL/min.
5
HPLC-based Quantification of BNB
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The measurement of BNB in each sample was carried out using high-performance liquid chromatography (HPLC) equipped with a fluorescence detector. The HPLC system consisted of a Chromatograph Waters Alliance 2695 and a Fluorescence Jasco FP-1520 detector that operated at an excitation wavelength of 310 nm and an emission wavelength of 390 nm. The chromatographic column was a Symmetry C18 (4.6 × 75 mm, 3.5 µm) and the mobile phase was Ammonium Formate 10 mM pH 7.4 (75:25 v/v); the flux was 1 mL/min and the volume of injection was 10 μL. The validated range for the quantification of BNB was from 0.03 to 1 µg/mL.
6
Quantification of Leaf Tocopherol Content
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Determinations were based in [65 (link)] and [23 (link)], with some changes for coffee leaves [49 (link)]. Briefly, 200 mg FW leaf tissue was homogenized in 3 mL of methanol, containing 0.24 mM of citric acid and 0.28 mM of isoascorbic acid, submitted to ultrasounds (5 min) and centrifuged (10,000 g, 5 min, 3°C). The supernatant was collected and the pellet was re-extracted, repeating the procedure twice. The supernatants were then combined, dried under vacuum, and the residue was re-suspended in 3 mL of acetonitrile, centrifuged (10,000 g, 3 min, 3°C) and filtered (PVDF, 0.45 μm), prior to a reversed-phase HPLC analysis, similar to that performed to ascorbate, except that a fluorescence detector (Jasco, FP1520, Japan, at 295 nm for excitation and 325 nm for detection) and methanol as eluent with a flow rate of 1 mL min-1, were used. TOC quantification was performed with a specific standard.
7
CSF Kynurenic Acid Quantification
8
HPLC Analysis of Urinary Biomarkers
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Urinary creatinine, neopterin, and uric acid concentrations were measured by the high-pressure liquid chromatography (HPLC) method, as previously described [58 (link)], by a Varian instrument (pump 240, autosampler ProStar 410) coupled to a UV–VIS detector (Shimadzu SPD 10-AV, λ = 240 nm for creatinine and uric acid; and JASCO FP-1520, λex = 355 nm and at λem = 450 nm) for neopterin).
After urine centrifugation at 13,000 rpm at 4 °C for 5 min, analytic separations were performed at 50 °C on a 5 µm Discovery C18 analytical column (250 × 4.6 mm I.D., Supelco, Sigma-Aldrich) at a flow rate of 0.9 mL/min. The calibration curves were linear over the range of 0.125–1 µmol/L, 0.625–20 mmol/L, and 1.25–10 mmol/L for neopterin, uric acid, and creatinine levels, respectively. The inter-assay and intra-assay coefficients of variation were <5%.
After urine centrifugation at 13,000 rpm at 4 °C for 5 min, analytic separations were performed at 50 °C on a 5 µm Discovery C18 analytical column (250 × 4.6 mm I.D., Supelco, Sigma-Aldrich) at a flow rate of 0.9 mL/min. The calibration curves were linear over the range of 0.125–1 µmol/L, 0.625–20 mmol/L, and 1.25–10 mmol/L for neopterin, uric acid, and creatinine levels, respectively. The inter-assay and intra-assay coefficients of variation were <5%.
9
Urinary Neopterin and Creatinine Analysis
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Urinary neopterin concentrations were measured by an isocratic HPLC method and were normalized to urine creatinine concentrations. Urine samples, stored at −20 °C were thawed and centrifuged at 13,000 rpm for 5 min at 4 °C; the supernatant was then adequately diluted with chromatographic mobile phase (15 mM of K2HPO4, pH 3.0). Neopterin and creatinine levels were measured using a Varian instrument (pump 240, autosampler ProStar 410) coupled to a fluorimetric detector (JASCO FP-1520, λex = 355 nm and at λem = 450 nm) for neopterin detection and to an ultraviolet-visible detector (Shimadzu SPD 10-AV, λ = 240 nm) for creatinine determinations. Analytic separations were performed at 50 °C on a 5 µm Discovery C18 analytical column (250 × 4.6 mm I.D., Supelco, Sigma-Aldrich, Bellefonte, PA 16823-0048, USA) at flow rate of 0.9 mL/min. The calibration curves were linear over the range of 0.125−1 μmol/L and of 1.25−10 mmol/L for neopterin and creatinine levels, respectively. Inter-assay and intra-assay coefficients of variation were <5%.
10
HILIC-FLD Analysis of 2-AA Glycans
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2-AA labelled glycan samples were detected by HILIC-FLD using an Azura HPLC system (Knauer Wissenschaftliche Geräte GmbH, Berlin, Germany) coupled to a Jasco FP-1520 fluorescence detector and a column thermostate (JASCO International Co., Ltd., Tokio, Japan) equipped with an OTU-Amino column (105Å, 5 µm, 250 mm × 4.6 mm; Applichrom GmbH, Oranienburg, Germany). A fluorescence excitation of 360 nm and emission of 428 nm was used to detect 2-anthranilic acid(2-aminobenzoic acid, 2-AA)-labeled glycans and dextran standards. Samples were resolved on HILIC at 40°C column temperature by isocratic elution with 60% acetronitrile in 10 mM ammonium formate buffer pH 4.4 at a flow rate of 1.0 ml/min.
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