Heparin was partially digested with heparinase I. The oligosaccharides were fractionated using a GE AKTA purifier 100 system with a Superdex TM Peptide 10/300 GL column. The fractions were eluted with 0.2 M NH4HCO3 at a flow rate of 0.4 ml/min. The tetrasaccharide fraction was then separated using a Spherisorb S5 SAX 5 μm column. A linear gradient of 0–2.0 M NaCl (pH 3.5) over 2 h was used. The flow rate was 4 ml/min. UV absorption was monitored at 232 nm. 1H spectra were used to characterize the purity based on the signal integration.
Akta purifier 100 system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The AKTA Purifier 100 system is a versatile and automated chromatography system designed for protein purification. It offers precise control of flow rate, pressure, and pH, enabling efficient separation and purification of a wide range of biomolecules. The system is equipped with advanced detection capabilities and is compatible with a variety of chromatography media and columns, making it a valuable tool for researchers and scientists in the field of biopharmaceutical development and life science research.
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Lab products found in correlation
C18 cartridge, by Merck Group (5 mentions)
Itraq reagent 8 plex multiplex kit, by AB Sciex (2 mentions)
Peptide 10 300 gl column, by GE Healthcare (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Modified lowry protein assay kit, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Capto core 700, by GE Healthcare (1 mentions)
Frac 950 fraction collector, by GE Healthcare (1 mentions)
Multiple affinity removal lc column human 14, by Agilent Technologies (1 mentions)
Itraq kit, by AB Sciex (1 mentions)
12 protocols using akta purifier 100 system
1
Fractionation of Heparin Oligosaccharides
2
Peptide Fractionation and Desalting
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The labeled peptides were pooled and fractionated on an AKTA Purifier 100 system (GE Healthcare) using a SCX column (PolyLCInc, Columbia, MD, United States). Briefly, the peptides were dried in a vacuum concentrator, dissolved in 2 mL of buffer A (10 mM KH2PO4 in 25% of ACN, pH 3.0) and loaded onto a 4.6 mm × 100 mm Polysulfoethyl column (5 μm, 200 A, PolyLC Inc.) at a flow rate of 1 mL/min. The peptides were eluted at a flow rate of 1 mL/min with a gradient of 0–10% buffer B (500 mM KCl, 10 mM KH2PO4 in 25% of ACN, pH 3.0) for 7 min, 10–20% buffer B for 10 min, 20–45% buffer B for 5 min, and 45–100% buffer B for 5 min. The eluted peptides were collected and desalted using an offline fraction collector. The collected fractions were combined into 15 pools, vacuum freeze-dried, and desalted on C18 cartridges (Sigma). All collected fractions were stored at –80°C until further analysis.
3
Proteomics Profiling of Major Depressive Disorder
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Before labeling, protein digestion for resultant peptide mixtures was performed according to the pooled sample preparation procedure described previously.20 (link) Briefly, the proteins (100 µg) in each sample were reduced, blocked on cysteine, alkylated and subsequently digested with trypsin overnight at 37°C. Prior to iTRAQ labeling, digested samples were desalted with a C18 cartridge (Sigma-Aldrich Co., St Louis, MO, USA; EMD Millipore, Billerica, MA, USA) to remove urea, salt and other reagents. Then, the purified sample was evaporated to dryness with a SpeedVac (RVT4104; Thermo Fisher Scientific, Waltham, MA, USA). After resuspension with 30 µL 0.5 M triethylammonium bicarbonate (pH 8.5), samples were labeled with iTRAQ reagents following the protocol provided by the manufacturer. Proteins obtained from control samples were labeled with iTRAQ Reagent-8plex Multiplex Kit (AB Sciex, Framingham, MA, USA), which contained 113, 114 and 115 reporter tags. Proteins obtained from patients with MDD were labeled with 116, 117 and 118 reporter tags. iTRAQ-labeled peptides were mixed and fractionated by SCX chromatography using the AKTA Purifier 100 system (GE Healthcare UK Ltd, Little Chalfont, UK). For each test, 33 SCX fractions were collected followed by a C18 cartridge (66872-U; Sigma-Aldrich Co.; EMD Millipore) for desalination.21 (link)
4
Antibody Production against OxyR Protein
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The oxyR gene was cloned by PCR from A. pleuropneumoniae 4074 using the primers P28oxyR-F/R, which contained NdeI and XhoI sites, respectively. The amplified fragment was ligated to pMD18-T for sequencing, digested with NdeI and XhoI, and ligated to digested pET28a (+) to construct the plasmid pET28a-oxyR. The pET28a-oxyR plasmid was transformed into E. coli BL21 (DE3) for expression. After cloning and successful expression, the rOxyR was purified by the AKTA Purifier 100 System (GE Healthcare, Bucks, United Kingdom) using a HisTrap FF affinity chromatography column.
Two male experimental Japanese big-eared white rabbits (about 2.5 kg, 8 weeks old) labeled as rabbit 1 and rabbit 2 were initially raised for a week. For immunization, rOxyR (1mg) antigen was mixed with Freund’s complete adjuvant (Sigma, St. Louis, Mo, USA) and injected subcutaneously into rabbits 1 and 2. After 12 days, a second immunization was administered using half the antigen volume and Freund’s incomplete adjuvant. Subsequent immunizations occurred every two weeks with the same antigen-adjuvant mixture. Blood (1 mL) was collected after each immunization for anti- rOxyR serum preparation, isolated a week after the final immunizations upon antibody detection. OxyR antiserum were purified by antigen affinity purification using the rOxyR.
Two male experimental Japanese big-eared white rabbits (about 2.5 kg, 8 weeks old) labeled as rabbit 1 and rabbit 2 were initially raised for a week. For immunization, rOxyR (1mg) antigen was mixed with Freund’s complete adjuvant (Sigma, St. Louis, Mo, USA) and injected subcutaneously into rabbits 1 and 2. After 12 days, a second immunization was administered using half the antigen volume and Freund’s incomplete adjuvant. Subsequent immunizations occurred every two weeks with the same antigen-adjuvant mixture. Blood (1 mL) was collected after each immunization for anti- rOxyR serum preparation, isolated a week after the final immunizations upon antibody detection. OxyR antiserum were purified by antigen affinity purification using the rOxyR.
5
H7N9 Virus Purification and Quantification
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The H7N9 bulks produced in sMDCK cells were purified according to a previous study [22 (link)] with the following modifications. First, 400 mL of the harvested virus was inactivated, and then the cell debris was removed using centrifugation at 1800×g for 30 min. Next, the inactivated virus was further purified using Capto Q and Capto core 700 anion exchange chromatography columns in an AKTA purifier 100 system (GE Healthcare). The flow-through virus solution was diafiltered with PBS using tangential flow filtration with a 100 kDa membrane cassette (Sartorius). Finally, this purified virus bulk was sterilized by using a 0.22 μm filter and stored at 4 °C. The amount of HA antigen in the bulk virus stock was calculated based on the band intensity of the viral protein on a 10% NuPAGE Bis-Tris gel (Thermo Fisher Scientific), and the amount of total viral protein was measured using a Modified Lowry Protein Assay kit (Thermo Fisher Scientific) [23 ].
6
Peptide Fractionation by SCX Chromatography
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All labeled peptides were mixed and fractionated using strong cation exchange chromatography and the AKTA Purifier 100 system (GE Healthcare, Milwaukee, WI, USA). The column was a 4.6 × 100 mm polysulfethyl column (5 μm, 200 Å) (PolyLCInc., Columbia, MD, USA), and buffer A (10 mM KH2PO4 pH 3.0 in 25% CAN), buffer B (500 mM KCl and 10 mM KH2PO4 pH 3.0 in 25% CAN). The chromatographic column was equilibrated with buffer A, and the sample was applied to the chromatographic column. The flow rate was set to 1 mL/min. The solvents were applied using the time gradient from 0 to 100% buffer B and followed by 60 min at 0%.
The absorbance values were monitored at 214 nm during elution, and the fractions were collected every 1 min. The samples were freeze-dried and then desalted using C18Cartridges (66872-U, Sigma, St. Louis, MO, USA). All samples were stored at −80°C until the liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis.
The absorbance values were monitored at 214 nm during elution, and the fractions were collected every 1 min. The samples were freeze-dried and then desalted using C18Cartridges (66872-U, Sigma, St. Louis, MO, USA). All samples were stored at −80°C until the liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis.
7
Strong Cation Exchange Fractionation Protocol
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All of the labeled peptides were mixed and then fractionated by strong cation exchange (SCX) chromatography using AKTA Purifier 100 system (GE Healthcare). The used column was Polysulfoethyl 4. 6 ×100 mm column (5 µm, 200 Å) (PolyLCInc, Maryland, USA), and solvent A was 10 mM KH2PO4 pH 3.0 in 25% of ACN, solvent B was 500 mM KCl, 10 mM KH2PO4 pH 3.0 in 25% of ACN. The solvents were applied using the time gradient from 0–100% solvent B and followed by 10 min at 0%. A total of 36 fractions were collected and finally combined into 15 pools. C 18Cartridges (66872-U, Sigma) was used to desalt the freeze-dried samples and all samples were stored at −80 °C until LC-MS/MS analysis.
8
Strong Cation Exchange Fractionation of iTRAQ-labeled Peptides
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The iTRAQ-labeled peptides were fractionated with strong cation exchange (SCX) chromatography using the AKTA Purifier 100 system (GE Healthcare, Chicago, IL). The vacuum-dried peptide mixture was reconstituted with buffer A (10 mM of KH2PO4 in 25% ACN, pH 3.0) and loaded onto a 4.6 × 100 mm PolySULFOETHYL column (5 μm, 200 Å; PolyLC Inc., Columbia, MD). Then, the peptides were eluted at a flow rate of 1 ml/min with a gradient of 0–8% buffer B (500 mM of KCl, 10 mM of KH2PO4 in 25% of ACN, pH 3.0) for 22 min, a gradient of 8–52% buffer B for 25 min, a gradient of 52–100% buffer B for 3 min, and a gradient of 100% buffer B for 8 min. The elution was detected by measuring the absorbance at 214 nm, and the fractions were collected every 1 min. A total of 30 fractions were collected, pooled, and desalted on C18 Cartridges (66872-U Sigma).
9
Purification of Sialidase Enzyme
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The culture supernatant was filtered through a 0.45-μm filter, concentrated, and then subjected to immobilized metal ion affinity chromatography (IMAC) using a nickel-nitrilotriacetic acid (Ni-NTA) column coupled to an AKTA purifier 100 system (GE Healthcare, Umeå, Sweden). The column was loaded with the sample and then washed with wash buffer (50 mM PBS, 50 mM imidazole, pH 8.0), to remove nonspecific bound proteins. The flow rate was maintained at a constant 1 mL/min. The column was then eluted with elution buffer (50 mM PBS, 100 mM imidazole, pH 8.0) to remove the bound SIase, which was then dialyzed against 20 mM pH 8.0 Tris-HCl buffer using a 10 kDa cut-off filter and then concentrated using ultrafiltration. The purified SIase was subjected to SDS-PAGE using 12% gel and its protein concentration was determined using the BCA method, with bovine serum albumin (BSA) as a standard.
10
Protein Separation by Size Exclusion Chromatography
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