Sargassum thunbergii (3.0 kg) was collected from Yantai, Shandong province (China). DEAE-cellulose 52 and Sephacryl S-300 HR were purchased from GE Healthcare Life Sciences (Shanghai, China). Other analytical chemical regents were supplied from Sinopharm (Shanghai, China).
Sephacryl s 300 hr
Manufactured by GE Healthcare
Sourced in United States
Sephacryl S-300 HR is a size-exclusion chromatography medium used for the separation and purification of macromolecules, such as proteins, nucleic acids, and polysaccharides, in aqueous solutions. It is composed of cross-linked copolymers of allyl dextran and N,N'-methylene bisacrylamide, which provide a porous structure for size-based separation.
Automatically generated - may contain errors
Lab products found in correlation
Colorimetric assay, by Abcam (3 mentions)
Deae sepharose fast flow deae qff, by GE Healthcare (2 mentions)
Deae 52 cellulose, by GE Healthcare (1 mentions)
Quikchange kit, by Agilent Technologies (1 mentions)
Nickel nitrilotriacetic acid, by Qiagen (1 mentions)
Hitrap q hp, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Monosaccharide standard, by Merck Group (1 mentions)
Q sepharose fast flow, by GE Healthcare (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Human plasminogen, by Merck Group (1 mentions)
Bovine serum albumin fraction 5, by Merck Group (1 mentions)
Low molecular weight marker protein, by GE Healthcare (1 mentions)
Akta purification system, by GE Healthcare (1 mentions)
D glucose, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
L arabinose, by Merck Group (1 mentions)
L rhamnose, by Merck Group (1 mentions)
D mannose, by Merck Group (1 mentions)
16 protocols using sephacryl s 300 hr
1
Extraction of Sargassum thunbergii Compounds
2
Purification and Characterization of KlUA Variants
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The KlUA gene was amplified from the Kluyveromyces lactis genome, and inserted into vector pET28A (Novagen). To generate the KlUAΔBCCP construct, a stop codon was inserted after base pair 5223. This plasmid was transformed into Escherichia coli BL21 (DE3) Star cells, which were cultured in LB medium supplemented with 50 mg/l kanamycin, and induced with 0.3 mM isopropyl β-D -1-thiogalactopyranoside when the optical density at 600 nm reaches 1.0, at 16°C for 16 hours. The KlUAΔBCCP protein was purified by nickel-nitrilotriacetic acid (Qiagen), anion-exchange (Hitrap Q HP, GE Healthcare) and size exclusion columns (Sephacryl S300 HR, GE Healthcare). Purified protein was concentrated to 10 mg/ml, in a buffer containing 20 mM Tris/HCl (pH 8.0), 200 mM NaCl, 2 mM DTT and 5% glycerol, flash cooled in liquid nitrogen and stored at −80°C.
The expression and purification of the full length KlUA, Kluyveromyces lactis UC (KlUC) and Kluyveromyces lactis AH (KlAH) followed protocols reported previously [14 (link),17 (link)].
The KlUA mutants were generated with the QuikChange kit (Agilent Technologies) and verified by DNA sequencing. The mutants were expressed and purified following the same protocol for the wild type protein.
The expression and purification of the full length KlUA, Kluyveromyces lactis UC (KlUC) and Kluyveromyces lactis AH (KlAH) followed protocols reported previously [14 (link),17 (link)].
The KlUA mutants were generated with the QuikChange kit (Agilent Technologies) and verified by DNA sequencing. The mutants were expressed and purified following the same protocol for the wild type protein.
3
Preparation of Fluorescent Lipid Vesicles
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1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-plamitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, l -α-phosphatidyl-inositol, cardiolipin, cholesterol and 18:1 DGS-NTA(Ni) were mixed at a weight ratio of 36:22:9:8:20:5, and dried as thin films in glass test tubes under nitrogen for 16 h. To encapsulate FITC-labeled dextran 10 (F-d10), 50 mg F-d10 was added to 50 mg lipid in 1 ml of 150 KCl buffer containing 20 mM HEPES (pH 7.0), sonicated and extruded 15 times through a 400 nm polycarbonate membrane. Liposomes with F-d10 were further purified by gel filtration on Sephacryl S-300 HR (GE Healthcare). Phosphate was determined by colorimetric assay (Abcam, Cambridge, UK).
4
Fabrication of Fe3O4@DMSA-Rituximab Nanoprobe
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The nanoparticles of Fe3O4@DMSA were coupled with antibody via covalent bond formation between the free carboxylic groups of DMSA and the amine groups of rituximab. EDC (10 µg/µL, 10 µL) and sulfo-NHS (10 µg/µL, 5 µL) were added to 0.5 mL of Fe3O4@DMSA aqueous (1 mg) solution in borate buffer (0.04 M, pH 9) to activate the carboxylic groups. The activation reaction continued for 30 minutes in a thermostat oscillator at 25°C. Rituximab was then added to the mixture with further oscillation for 2 hours. The Fe3O4@DMSA@Ab nanoprobe solution was purified by gel chromatography (Sephacryl S-300 HR; GE Healthcare, Chicago, IL, USA) and the initial 1 mL from percolate of the Fe3O4@DMSA@Ab nanoprobes was collected. The remaining filtrates were carefully collected and tested to determine the rituximab content in the Fe3O4@DMSA@Ab sample (Figure S1B–D ). After adding 110 µL aqueous BSA solution (10%), the Fe3O4@DMSA@Ab solution was stored at 4°C for follow-up experiments. The following percolates were collected to detect the amount of un-coupled antibodies using the BCA assay.
5
Structural Analysis of Durvillaea antarctica
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Dry brown algae (Durvillaea antarctica) was provided by Gather Great Ocean Algae industry group CO., LTD (Qingdao, China). The raw material was milled into a powder and stored in a dry environment at room temperature. Q Sepharose Fast Flow and Sephacryl S-300/HR were purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA). Pullulan standards for HPGPC analysis were purchased from Showa Denko K.K. (Tokyo, Japan). Monosaccharide standards for sugar composition analysis were purchased from Sigma (St. Louis, MO, USA). Pyromellitic acid (PMA), sodium fluoride (NaF), methyl iodide (CH3I), NaH, and KBr were also obtained from Sigma. Other materials, such as 732 cation-exchange resin, pyridine, and dimethylsulfoxide (DMSO), were from Signopharm Chemical Reagent CO., LTD (Shanghai, China).
6
Preparation and Characterization of DLBS1033
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DLBS1033 was prepared at Dexa Laboratories of Biomolecular Sciences (Cikarang, Indonesia) as described previously.1 (link) Bovine serum albumin (BSA) fraction V, human plasminogen, and thrombin for biochemistry were purchased from Merck KGaA (Darmstadt, Germany). Human fibrinogen fraction I-S and fetal calf serum were obtained from Sigma-Aldrich Corp (St Louis, MO, USA). Low-molecular-weight marker protein and Sephacryl® S-300HR were purchased from GE Healthcare (Little Chalfont, UK). Na99mTcO4 was obtained from National Nuclear Energy Agency (Tangerang, Indonesia). Other reagents used were of analytical grade.
7
Purification of Ovalbumin from Egg White
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OVM was crude extracted according to a previously reported method [21 (link)] with modifications. In brief, 200 mL of fresh whole egg white was stirred at 4 °C for 30 min and subsequently diluted with 600 mL of 100 mM NaCl. The pH was adjusted to 6.0 with 1 M HCl, and the solution was incubated overnight at 4 °C. The egg white solution was centrifuged at 10,000g for 10 min at 4 °C, and the precipitate was resuspended with 500 mM NaCl for 4 h and then centrifuged under the same conditions.
After the precipitate, which was crude OVM, was washed several times with distilled water, the above procedure was repeated. The extract was freeze-dried and stored at −20 °C. OVM crude extract was purified by gel filtration chromatography (Sephacryl S-300 HR, 26 mm × 60 cm) using the AKTA purification system (GE, USA). The target elution peak was dialyzed by distilled water, and the product was purified OVM.
After the precipitate, which was crude OVM, was washed several times with distilled water, the above procedure was repeated. The extract was freeze-dried and stored at −20 °C. OVM crude extract was purified by gel filtration chromatography (Sephacryl S-300 HR, 26 mm × 60 cm) using the AKTA purification system (GE, USA). The target elution peak was dialyzed by distilled water, and the product was purified OVM.
8
Active Compound Purification Protocol
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Adsorption chromatography. The primary enrichment of the active compound/s was achieved by adsorption chromatography on a polystyrene resin (Amberlite XAD-2; Rohm and Haas, Philadelphia, Pa.). The resin (4 g) was placed in a glass column (11 cm by 1 cm). The column was equilibrated Gut medium and then 40 mL of retentate fraction (SNC_TAE2020) was applied at a flow rate of approximately 1 mL min−1. The column was then washed with 3-bed volumes of Gut. The elution of the active compound/s was subsequently carried out with methanol and NaOH (0.5M) in methanol. Fractions of 30 mL were collected. The unbound fractions and the fractions eluted during the washing were concentrated (ten-times), while those eluted with methanol and NaOH in methanol were recovered, dried, to obtain more concentrated samples, and resuspended in a small volume (5 mL) of Gut medium. Each chromatographic fraction was analyzed by surface coating assay against S. epidermidis O-47 and S. epidermidis RP62A.
Gel filtration. The SNC_TAE2020 was dialyzed against Milli-Q water using a centricon cut-off 30kDa (Merck KGaA, Darmstadt, Germany). After the dialysis, the concentrated supernatant was further purified on a Sephacryl S-300HR (GE Healthcare Life Sciences, 0.5 × 110 cm, flow rate 15.6 mL h−1, fraction volume 2.5 mL) eluted with 0.05 M ammonium hydrogen carbonate.
Gel filtration. The SNC_TAE2020 was dialyzed against Milli-Q water using a centricon cut-off 30kDa (Merck KGaA, Darmstadt, Germany). After the dialysis, the concentrated supernatant was further purified on a Sephacryl S-300HR (GE Healthcare Life Sciences, 0.5 × 110 cm, flow rate 15.6 mL h−1, fraction volume 2.5 mL) eluted with 0.05 M ammonium hydrogen carbonate.
9
Extraction and Characterization of Dendrobium Polysaccharides
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Dendrobium nobile Lindl. was purchased from Guizhou Chishui Guo Li Dendrobium Development Co., Ltd. (Chishui, China). After drying and crushing, it was sieved through 80 meshes and left on standby. Ten kinds of standard monosaccharides, such as mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose, were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). ELISA kits (including TNF-α, IL-1β, IL-6 and IL-10) were purchased from Shanghai Jianglai Biological Co., Ltd. (Shanghai, China). DEAE-Sepharose Fast Flow (DEAE-QFF) and Sephacryl S-300 HR were purchased from GE Healthcare. Other chemicals and reagents were of analytical grade.
10
Isolation and Characterization of Pullulan from Coastal Seaweed
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M. angicava was collected from the coastal area of the Yellow Sea in Qingdao, China on June 2013, which is in the growth mature period of the seaweed. The sample is green and 4–9 cm in size. After washing thoroughly in sea water, the sample was dried. Pullulan standards (Mw: 5.9 kDa, 9.6 kDa, 21.1 kDa, 47.1 kDa, 107 kDa, 200 kDa, 344 kDa, and 708 kDa) were purchased from Showa Denko K.K. (Tokyo, Japan). Sephacryl S-400/HR and Sephacryl S-300/HR were purchased from GE Health care Life Sciences (Piscataway, NJ, USA). Dialysis membrane (molecular weight cut-off 1000, 3500) was purchased from Lvniao (Yantai, China). l -rhamnose, l -arabinose, d -xylose, d -mannose, d -galactose, d -glucose, d -glucuronic acid, and heparin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). APTT, TT and PT assay reagents were from MD Pacific (Tianjin, China). An FDP kit was purchased from BIOLINKS CO., LTD. (Tokyo, Japan). PAI-1 and D-dimer kits were purchased from Simens Healthcare Diagnostics Products (Marburg, Germany).
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