Release of haeme from wild-type NdCld and variants was tested by incubation of the respective proteins with horse heart apo-myoglobin (Sigma) [5 ,34 ]. Apo-myoglobin was prepared using a modified extraction method by Teale [35 ] as described previously [5 ]. Thirty micromolars of NdCld samples were incubated with 40 μM apo-myoglobin in 50 mM phosphate buffer, pH 7.0, for 1 h at room temperature. Size-exclusion chromatography was performed to separate NdCld and myoglobin. HPLC (Shimadzu prominence LC20) was equipped with a refractive index detector (RID-10A, Shimadzu), a diode array detector (SPD-M20A, Shimadzu) and a MALLS-detector (Multi-Angle-Laser-Light-Scattering; WYATT Heleos Dawn8+ plus QELS; software ASTRA 6). The column (Superdex 200 10/300 GL, GE Healthcare) with particle size of 13 μm was equilibrated with the running buffer (Dulbecco PBS, 200 mM NaCl). The experiments were performed at a flow rate of 0.75 ml·min−1, 80 μl of the protein solution (containing 30 μM NdCld and 40 μM apo-myoglobin) were injected. Untreated NdCld samples (wild-type, K141E, E210A, R173Q/E210A), holo- and apo-myoglobin as well as Bio-Rad Gel Filtration Standard (#151-1901) containing also holo-myoglobin from horse heart, were injected as references. Haeme transfer was observed by extracting elution profiles at 280 and 409 nm from the diode array detector.
Lc 20
Manufactured by Shimadzu
Sourced in Japan, United States
The Shimadzu LC-20 is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It features a dual-plunger, high-pressure pump for precise and reliable solvent delivery. The LC-20 is capable of operating at pressures up to 40 MPa, making it suitable for a wide range of analytical separations.
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Lab products found in correlation
Spd m20a, by Shimadzu (4 mentions)
Ascentis express c 18 column, by Shimadzu (3 mentions)
Rid 20, by Shimadzu (3 mentions)
Qtrap 5500 mass spectrometer, by AB Sciex (3 mentions)
Rid 10a, by Shimadzu (2 mentions)
Spd 20a, by Shimadzu (2 mentions)
Lcms 2020, by Shimadzu (2 mentions)
Luna c18, by Phenomenex (2 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
Prelude peptide synthesizer, by Protein Technologies (1 mentions)
Avance 3 600m, by Bruker (1 mentions)
Nicolet is10, by Thermo Fisher Scientific (1 mentions)
Lc 2000plus, by Jasco (1 mentions)
Gc 8a, by Shimadzu (1 mentions)
Phytic acid, by Merck Group (1 mentions)
Wheat phytase, by Merck Group (1 mentions)
Roa organic acid h 8 column, by Phenomenex (1 mentions)
Nicolet 6700 infrared spectrometer, by Thermo Fisher Scientific (1 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
71 protocols using lc 20
1
NdCld Haeme Transfer Assay
2
Polyphenolic Fingerprinting via HPLC
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High-pressure liquid chromatography (HPLC) was used to evaluate the polyphenolic fingerprinting of the extract as described above [50 (link)]. HPLC-DAD analyses were performed using a Shimadzu LC 20 (Kyoto, Japan), which was equipped with a diode array detector (DAD) and with a 150 × 4.6 mm i.d., 2.7 μm Ascentis Express C 18 column. The mobile phases were as follows: H2O/H3PO4 (99:1, solvent A), MeOH/CAN/H3PO4 (49,5:49,5:1, solvent B). The gradient used was as follows: concentration of the solvent A of 95% going to 77% (34 min), maintained at 77% (3 min), 74% (60 min), 60% (85 min), 20% (90 min), and 0% (92 min). The total time was 105 min. The column temperature was maintained at 25 °C. The flow was 1 mL/min, and the injection volume was 5 μL. The chromatogram profiles were recorded from 190 to 500 nm and monitored at 280 and 330 nm ± 2 nm.
3
Solid-phase Peptide Synthesis and Purification
4
Polymer Characterization via Spectroscopy
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Gel permeation
chromatography (GPC) was performed on water solutions of the sample
using a chromatograph (LC 20, Shimadzu, Japan). Fourier transform
infrared (FTIR) spectra were recorded with a spectrometer (Nicolet
IS 10, Thermo Fisher, America) over the 4000–400 cm–1 range, with a resolution of 0.4 cm–1. Solid-state 13C NMR spectroscopy and liquid-state 1H NMR spectroscopy
of PFA were conducted with a spectrometer (Bruker AVANCE III 600 M,
Karlsruhe, Germany).
chromatography (GPC) was performed on water solutions of the sample
using a chromatograph (LC 20, Shimadzu, Japan). Fourier transform
infrared (FTIR) spectra were recorded with a spectrometer (Nicolet
IS 10, Thermo Fisher, America) over the 4000–400 cm–1 range, with a resolution of 0.4 cm–1. Solid-state 13C NMR spectroscopy and liquid-state 1H NMR spectroscopy
of PFA were conducted with a spectrometer (Bruker AVANCE III 600 M,
Karlsruhe, Germany).
5
Determining CPPS Molecular Weight Using GPC
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Weight average molecular weight (Mw) of CPPS and sub-fractions were performed according to the report described by Li [37 (link)]. PEO standards were used to establish a calibration curve. The analysis was conducted on a gel permeation chromatography system (Shimadzu LC20, Shimadzu Co., Kyoto, Japan) equipped with a refractive index detector (Shimadzu RID-20, Shimadzu Co., Kyoto, Japan). The samples were loaded into a TSK Ultrahydrogel™ linear column (8 × 300 mm) coupled with a TSK Ultrahydrogel™ guard column (6 × 200 mm), and the column temperature was set at 35 °C. The 0.1 N nano3 solution containing 0.06% (w/v) nan3 was used as the mobile phase at the flow rate of 0.6 mL/min and the injection volume was 20 μL. Data was collected by Shimadzu labsolutions HW200 workstation.
6
Physiological Impacts on Microbial Metabolism
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All physiological experiments were performed in triplicate. The effects of temperature, pH, and concentrations of NaCl on cell growth, utilization of carbohydrates and sensitivity to antibiotics were determined by hydrogen production in gas phase of cultures. Hydrogen and methane in gas phase of cultures were measured with a gas chromatography equipped with a thermal conductivity detector (GC-8A; Shimadzu, Japan). Glucose, acetate, and ethanol in liquid phase of cultures were measured with a high-performance liquid chromatography (HPLC) (LC20; Shimadzu) with Shim-pack SPR-H column (Shimadzu) or HPLC (LC-2000Plus, Jasco) with Aminex HPX-87H column (BIO-RAD). Methyl esters of cellular fatty acids were identified and quantified via a gas chromatography-mass spectrometry (M7200A GC/3DQMS system; Hitachi).
7
Enzymatic Phytic Acid Hydrolysis
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Enzymatic hydrolysis of phytic acid (Sigma, Poznań, Poland) was performed as described elsewhere [14 (link)] with modifications. The reaction was conducted in a total volume of 5 mL containing 100 mg of phytate and 50 mg of wheat phytase (Sigma, Poznań, Poland) at a pH of 5.15 and a temperature of 55 °C. The reaction was stopped after two hours by heating (100 °C for 5 min). Next, the obtained hydrolysate of phytic acid (hPA120) was cooled down on ice and precipitated enzymatic proteins were centrifuged (12,000× g, 10 °C, 10 min). The supernatant was collected, filter sterilized (filter pore 0.22 µm), aliquoted, and stored at −20 °C. Directly before use, the hydrolysate was defrosted and diluted twice with McCoy 5A medium without antibiotics. Before applying onto colonocytes, the hPA120 was 10-fold diluted in a medium appropriate for investigated cell lines.
The composition of inositol phosphates in phytate hydrolysate was determined by applying HPLC-MS (LC-20, Shimadzu, Kyoto, Japan; QTRAP 5500 mass spectrometer, AB SCIEX, Vaughan, Canada) and identified using real standards comprised the retention time and the presence of the respective parent and daughter ion (negative) pairs (Table S1 ) [15 (link)].
The composition of inositol phosphates in phytate hydrolysate was determined by applying HPLC-MS (LC-20, Shimadzu, Kyoto, Japan; QTRAP 5500 mass spectrometer, AB SCIEX, Vaughan, Canada) and identified using real standards comprised the retention time and the presence of the respective parent and daughter ion (negative) pairs (
8
Comprehensive Characterization of EPS-160
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The total polysaccharide content of EPS-160 was measured by the phenol-sulfuric acid (H2SO4) method (Yin et al., 2014 (link)). The presence of protein was determined using the Bradford method (Blum et al., 2008 (link)). For monosaccharide composition analysis, 20 mg EPS-160 was hydrolyzed with 2 mL of 2 M trifluoroacetic acid (TFA) at 120°C for 4 h. The hydrolyzate was evaluated by high-performance liquid chromatography (HPLC) (Agilent1200, United States) equipped with a ROA-Organic Acid H+(8%) column (Phenomenex, United States) (Zhang et al., 2017 (link)). Functional groups of EPS-160 were identified by measuring from 400 to 4000 cm–1 wavenumbers in a Nicolet 6700 infrared spectrometer (Thermo Fisher, America). Molecular weights (MWs) of EPS-160 were analyzed by a gel permeation chromatography (GPC) system (LC20, Shimadzu, Japan). The system was equipped with a refractive index (RI) detector (RID-20, Shimadzu, Japan) and a TSK G4000PWXL column (Tosoh, Japan). The MWs was calculated according to the weight-averaged molecular weight.
9
HPLC-DAD Polyphenolic Fingerprinting
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The polyphenolic fingerprinting of the extract was defined by HPLC-DAD. The analyses were performed as described in Malfa et al. 2020 [44 (link)]. Specifically, 5 µL of dimethylformamide/water (9:1) sample solution (10 mg/mL) was analyzed in duplicate by HPLC-DAD using a Shimadzu LC 20 (Kyoto, Japan), equipped with a diode array detector (DAD) and with a 150 × 4.6 mm i.d., 2.7 µm Ascentis Express C 18 column maintained at 25 °C with a flow of 1 mL/min. Two mobile phases: H2O/H3PO4 (99:1, solvent A) and MeOH/ACN/H3PO4 (49.5:49.5:1 solvent B), were used with the following gradient: concentration of solvent A of 95% going to 77% (34 min.), maintained at 77% (3 min.), 74% (60 min.), 60% (85 min.), 20% (90 min.) and 0% (92 min.); total time 105 min. The chromatogram was recorded at 330 nm ± 2 nm.
10
In Vitro Drug Release Study in Simulated Lung Fluid
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For in vitro drug release study, the simulated lung fluid (SLF) was used to simulate the physiological environment in lungs, which was composed of 0.02% DPPC, 0.02% potassium dihydrogen phosphate, 0.318% disodium hydrogen phosphate, 0.02% potassium chloride, 0.01% magnesium chloride, 0.01% calcium chloride, and 0.80% sodium chloride (all w/v)45 (link). Samples containing 1 mg of ketoprofen and 1 mL of SLF were enclosed in the dialysis bags and incubated in 30 mL of SLF. After incubation in a constant temperature shaker (100 rpm, 37 °C), 1 mL of the release medium was withdrawn at predetermined time points, and then equivalent of fresh release medium was compensated for sample losing.
The obtained release medium was filtered through 0.22 μm membrane and analyzed by a high performance liquid chromatography system (HPLC, LC-20, Shimadzu, Japan). The mobile phase consisted of methanol and 0.02 mol/L monopotassium phosphate buffer solution (80:20, v/v) with a flow rate of 1 mL/min. Twenty microliters of the release medium were injected into a C18 column (5 μm, 250 mm × 4.6 mm, Diamonsil, Beijing, China), and detected at 258 nm. The column temperature was set at 40 °C.
The obtained release medium was filtered through 0.22 μm membrane and analyzed by a high performance liquid chromatography system (HPLC, LC-20, Shimadzu, Japan). The mobile phase consisted of methanol and 0.02 mol/L monopotassium phosphate buffer solution (80:20, v/v) with a flow rate of 1 mL/min. Twenty microliters of the release medium were injected into a C18 column (5 μm, 250 mm × 4.6 mm, Diamonsil, Beijing, China), and detected at 258 nm. The column temperature was set at 40 °C.
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