A total of 500 μL of appropriately diluted hydrolysates from filter paper was analyzed on a Thermo Scientific Dionex ICS-5000 high-performance liquid chromatography (Dionex Corporation, Sunnyvale, CA, United States) instrument equipped with a CarboPac PA100 guard column (4 × 50 mm) and an analytical column (4 × 250 mm) with a flow rate as 1 mL/min at 22°C. The samples were resolved in a mobile phase of 100 mM NaOH. Glucose and cellobiose were used as standards.
Pa100
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PA100 is a compact and portable pH/ion meter designed for versatile laboratory and field applications. It features a large, easy-to-read display and simple user interface for accurate pH, mV, and ion concentration measurements. The PA100 is a reliable and accurate instrument for a variety of testing and monitoring tasks.
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Lab products found in correlation
Dionex ics 5000, by Thermo Fisher Scientific (2 mentions)
Dnaengine thermo cycler, by Bio-Rad (1 mentions)
System gold hplc system, by Beckman Coulter (1 mentions)
Hpx 87h column, by Aminex (1 mentions)
Ltq xl linear, by Thermo Fisher Scientific (1 mentions)
Ics 3000, by Thermo Fisher Scientific (1 mentions)
Kestose, by Merck Group (1 mentions)
Nystose, by Merck Group (1 mentions)
Pegfp c1 vector, by Takara Bio (1 mentions)
8 protocols using pa100
1
Carbohydrate Analysis by HPLC
2
Enzymatic Activity of Recombinant UGMs
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The enzymatic activity of recombinant
UGMs was tested by monitoring the formation of UDP-Galp from UDP-Galf by HPLC. The assay was performed
in 0.1 mL of 25 mM HEPES, 125 mM NaCl, 20 mM dithionite, at pH 7.5,
at various concentrations of UDP-Galf. The reaction
was initiated by addition of enzyme at 50 nM for wild-type AfUGM,
1–3 μM for AfUGM mutants, 100 nM for wild-type TcUGM,
0.5–3 μM for TcUGM mutants, 15 nM for wild-type MtUGM,
and 500 nM for the MtUGMH68A. The reaction was incubated at 37 °C
until ∼30% conversion of UDP-Galf to UDP-Galp was achieved. The reaction was terminated by heat denaturation
at 95 °C for 5 min, in a DNA engine thermocycler (BioRad, Hercules,
CA). After centrifugation, the resulting mixture was injected onto
a PA-100 (Dionex) HPLC column. The sample was eluted isocratically
with 75 mM KH2PO4, pH 4.5, at 0.80 mL/min. Absorbance
at 262 nm was monitored to identify fractions containing substrate
and product. Under these conditions, UDP-Galp eluted
at 27.35 min and UDP-Galf at 34.19 min. The extent
of conversion was determined by comparing the integration of the substrate
and product peaks. The initial velocity data was fit to the Michaelis–Menten
equation to obtain the kcat and KM values.
UGMs was tested by monitoring the formation of UDP-Galp from UDP-Galf by HPLC. The assay was performed
in 0.1 mL of 25 mM HEPES, 125 mM NaCl, 20 mM dithionite, at pH 7.5,
at various concentrations of UDP-Galf. The reaction
was initiated by addition of enzyme at 50 nM for wild-type AfUGM,
1–3 μM for AfUGM mutants, 100 nM for wild-type TcUGM,
0.5–3 μM for TcUGM mutants, 15 nM for wild-type MtUGM,
and 500 nM for the MtUGMH68A. The reaction was incubated at 37 °C
until ∼30% conversion of UDP-Galf to UDP-Galp was achieved. The reaction was terminated by heat denaturation
at 95 °C for 5 min, in a DNA engine thermocycler (BioRad, Hercules,
CA). After centrifugation, the resulting mixture was injected onto
a PA-100 (Dionex) HPLC column. The sample was eluted isocratically
with 75 mM KH2PO4, pH 4.5, at 0.80 mL/min. Absorbance
at 262 nm was monitored to identify fractions containing substrate
and product. Under these conditions, UDP-Galp eluted
at 27.35 min and UDP-Galf at 34.19 min. The extent
of conversion was determined by comparing the integration of the substrate
and product peaks. The initial velocity data was fit to the Michaelis–Menten
equation to obtain the kcat and KM values.
3
Enzymatic Synthesis and Purification of 2-5A
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2–5A (p3(A2′p)nA, where n = 1 to ≥3) was enzymatically synthesized from ATP using hexahistidine-tagged and purified recombinant porcine 42-kDa 2-5A synthetase (OAS1). Individual 2-5A oligomers were purified (>95% purity) using a Dionex PA100 (22 mm × 250 mm) semi-preparative column interfaced with a Beckman system gold HPLC system under the control of 32-Karat workstation [62 (link)]. The 5’-triphosphorylated triadenylate, p3A2’p5’A2’p5’A, was used for RNase L activation. Biotinlyation of 2-5A was performed using periodate chemistry as described earlier [30 (link), 44 (link)].
4
HPLC Analysis of Monosaccharide Decomposition
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Monosaccharides (D-xylose, D-glucose, and D-fructose) were determined using a high-performance liquid chromatography (HPLC) system Dionex 5,000 with acarbo Pac PA-100 (4 × 250 mm) anion exchange column and pulsed amperometric detection. Four different eluents were used: (A) 100 mM NaOH, (B) 100 mM NaOH + 600 mM NaCl, (C) 500 mM NaCl, and (D) ultra-deionized water. The decomposition products were analyzed using high performance liquid chromatography coupled with UV detection at 210 nm for organic acids and 284 nm for furanic compounds. 5 mM sulfuric acid was used as the elution solvent with a flow rate of 0.6 mL/min on a Aminex HPX-87H column (300 × 7.8 mm) at 45°C (Istasse et al., 2018 (link)).
The yield of all the compounds were defined by calculating the molar ratio between the generated product and the initial monosaccharides concentration (Figure S2 ).
The yield of all the compounds were defined by calculating the molar ratio between the generated product and the initial monosaccharides concentration (
5
Oligosaccharide Profiling by HPAEC-PAD and ESI-MS
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The OGs produced by overnight pectin-treated mutants were qualitatively characterized by the High-Performance Anion Exchange Chromatography-Pulsed Amperometric Detector (HPAEC-PAD) method using an analytical PA100 column (4 × 250 mm) connected to an analytical Dionex ICS-3000 ion chromatography system. Proteins must be removed by filtration through a 0.22 µm hydrophilic membrane before HPAEC-PAD analysis. The samples were eluted at a flow rate of 0.3 mL/min using the following protocol: 0-10 min, equilibrate the columns with 90% Eluent A (400 mM NaOH) and 10% Eluent B (400 mM NaOH, 1 M NaAc); 10-80 min, separate OGs with eluent A from 90 to 20% and eluent B from 10 to 80%; 80-90 min, clean the column with 100% eluent B.
Electrospray Ionization Mass Spectrometry (ESI-MS) was used to further determine the composition and the degree of polymerization (DP) of the product. After centrifugation, a 1 µL supernatant was injected into an LTQ XL linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Oligosaccharides were detected in negative ion mode with the following settings: ion source voltage 4.5 kV; capillary temperature 275-300°C; tube lens, 250 V; sheath gas, 30 arbitrary units (AU); scan mass range 150-2000 m/e.g.
Electrospray Ionization Mass Spectrometry (ESI-MS) was used to further determine the composition and the degree of polymerization (DP) of the product. After centrifugation, a 1 µL supernatant was injected into an LTQ XL linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Oligosaccharides were detected in negative ion mode with the following settings: ion source voltage 4.5 kV; capillary temperature 275-300°C; tube lens, 250 V; sheath gas, 30 arbitrary units (AU); scan mass range 150-2000 m/e.g.
6
Anion Exchange Chromatography for DMB Analysis
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Separation was achieved using a DNAPac PA-100 analytical anion exchange column (25 cm x 4 mm, packed with 0.1 µM microbeads, Dionex, UK). DMB fluorescence was monitored with a fluorescence detector (RF-10A) as described above. The mobile phases used were: distilled water (mobile phase A) and ammonium acetate buffer (5 M, pH 7.4, mobile phase B) for 30 minutes and 1 ml/min flow rate, with the gradient outlined in Supplementary Data, Table 1.
7
Profiling Fructans by Ion-Exchange Chromatography
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The distribution profiles of fructans were carried out in a liquid ion-exchange chromatograph DIONEX ICS-5000 (Thermoscientific, Waltham, MA, USA), using a Carbopac PA-100 (40 × 250 mm) column with a precolumn (40 × 25 mm) with a gradient of 100 mM NaOH and 600 mM CH3COONa for 90 minutes at 35 °C. Chicory inulin (SIGMA ALDRICH I-2255, St. Louis, MO, USA) was used as a standard. Additionally, standards of glucose, fructose, sucrose, kestose and nystose (SIGMA ALDRICH, St. Louis, MO, USA) were included to complete the distribution profile. Prior to their injection, samples were diluted in mili-Q water (2 mg·mL−1) and filtered through a 0.45 µm (Millipore®, Burlington, MA, USA) nylon membrane; the obtained results were expressed in nC versus time.
8
Expression of Epitope-Tagged DPR and DDX Proteins
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cDNAs encoding FLAG-tagged GA100, GR100, PR100, PA100, or PR5 were synthesized (Thermo Fisher Scientific, Waltham, MA, USA) and subcloned into pEF1/myc-His vector (Thermo Fisher Scientific) or pEGFP-C1 vector (Takara, Shiga, Japan) to express FLAG-tagged or EGFP-FLAG-tagged DPR proteins, respectively. A stop codon was inserted just after the DPR protein-encoding sequence. Human DDX17 and DDX18 cDNAs were amplified from human adult normal testis cDNA (BioChain Institute, Newark, CA, USA). Human DDX21 cDNA were amplified from HeLa cell cDNA. These DDX-encoding cDNAs were subcloned into pEF4/His vector (Thermo Fisher Scientific) in which HA tag-encoding sequence was inserted before Xpress tag-encoding sequence to express HA-tagged DDX17, DDX18, and DDX21. Human DDX5-HA-myc-His6-encoding plasmid was kindly provided by Dr. Didier Auboeuf (Université de Lyon)51 (link). Human Myc cDNA was amplified from human adult normal testis cDNA (BioChain Institute).
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