PLN’s size and size distribution were determined by dynamic light scattering (DLS) measurements using a Malvern instrument (NANO ZS, Malvern Instruments, CA, USA). The Z-average particle size was measured in triplicate. The integrity of the radiolabeled PLN was determined by several analytical methods. The samples were assessed by size exclusion LPLC equipped with a size exclusion Sepharose CL 6B column (25 × 300 mm, GE healthcare, 1.5% NaCl, pH 6.8;0.8mL/min)(modified for pressure pump use), an UV monitor, a fluorescent monitor and an on-line flow radioactivity detector (Bioscan Inc., Washington, DC, USA)(Lee et al., 2013b (link)). The radiochemical purity of 99mTc-PLN was also evaluated by ascending ITLC (Gelman Sciences, Inc., Ann Arbor, MI, USA) using 100% acetone as the solvent phase (Arulsudar et al., 2003 (link)). Calcein entrapment efficiency was determined by fluorescent intensity change at 495nm Excitation (Ex)/ 515nm Emission (Em), using a fluorescence microplate reader (SpectraMax M2, Molecular Devices) after adding the detergent Triton X 100 (TX100, 0.02% final concentration). The drug encapsulation efficiency was calculated using the following formula: Ft/F0 where Ft is the fully dequenched drug fluorescence intensity after the addition of TX100 and F0 is the fluorescence intensity of drug before the addition of TX100.
Sepharose cl 6b column
Manufactured by GE Healthcare
Sourced in United States
Sepharose CL-6B is a size-exclusion chromatography medium used for the separation and purification of biomolecules such as proteins, nucleic acids, and other macromolecules. It is a cross-linked agarose-based matrix with a defined pore size distribution, providing effective fractionation of samples within a specific molecular weight range.
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Lab products found in correlation
Nano z, by Malvern Panalytical (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
On line flow radioactivity detector, by Bioscan (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Pectin, by Merck Group (1 mentions)
Pectinase, by Merck Group (1 mentions)
Phastsystem, by GE Healthcare (1 mentions)
Laborota 4000, by Heidolph (1 mentions)
6 protocols using sepharose cl 6b column
1
Characterization of Radiolabeled Lipid Nanoparticles
2
Purification and Characterization of HBHA Protein
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The positive colonies resistant to 5 mg/ml G418 were inoculated in 100 ml of BMGY at 30°C with constant shaking at 250 rpm until the optical density at 600 nm reached 3.0. The sediment of P. pastoris was resuspended in 20 ml of BMMY, and continuously induced for 96 hrs at 30 °C with shaking at 250 rpm. Methanol was maintained at a concentration of 1% (v/v). Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to confirm that HBHA was expressed successfully.
The supernatant was separated from the culture, and the purification was completed using a Sepharose CL-6B column (GE Healthcare, Somerset, NJ, USA). HBHA protein was lyophilized, diluted in phosphate-buffered saline (PBS) using pyrogen-free reagents, aliquoted, and stored at −20°C. The protein concentration was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). The purified protein was confirmed by western blotting with anti-HBHA protein mouse serum (diluted 1/800) as the primary antibody and peroxidase-conjugated goat anti-mouse IgG (diluted 1/5000; Proteintech Biotech, Wuhan, China) as the secondary antibody. The immunoblots were visualized using enhanced chemiluminescence technology (Tiangen Biotech, China).
The supernatant was separated from the culture, and the purification was completed using a Sepharose CL-6B column (GE Healthcare, Somerset, NJ, USA). HBHA protein was lyophilized, diluted in phosphate-buffered saline (PBS) using pyrogen-free reagents, aliquoted, and stored at −20°C. The protein concentration was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). The purified protein was confirmed by western blotting with anti-HBHA protein mouse serum (diluted 1/800) as the primary antibody and peroxidase-conjugated goat anti-mouse IgG (diluted 1/5000; Proteintech Biotech, Wuhan, China) as the secondary antibody. The immunoblots were visualized using enhanced chemiluminescence technology (Tiangen Biotech, China).
3
Pectin Extraction and Purification
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Pectin (from apple) and Pectinase (from Aspergillus niger) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The ramie fiber was purchased from a local vendor, Gwangju, South Korea. The Sepharose CL-6B Column was obtained from GE Healthcare Bio-Science AB (Uppsala, Sweden). All analytical grade reagents were used.
4
Purification of Adenia heterophylla Lectin
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Adenia heterophylla caudex was purchased from Exotica Botanical Rarities, Erkelenz-Golkrath, Germany, and was kept in the greenhouse of the Botanical Garden of the University of Bologna until use. A. heterophylla caudex (460 g) was decorticated and homogenized, as described in [27 (link)]. The supernatant (300 mL, containing 650.2 mg of protein) was loaded onto a Sepharose CL-6B column (30 cm height × 2.6 cm diameter) (GE Healthcare, Buckinghamshire, UK), pre-treated with 0.2 M HCl, as described in [27 (link)]. After washing with PBS to remove the unbound substances, the lectin proteins, bound to the resin, were eluted stepwise, using 0.5 M galactose in PBS. The protein content of crude extract and not retained material was spectrophotometrically determined, using the Kalb and Bernlohr method [53 (link)].
The fractions eluted from Sepharose CL-6B were analyzed by SDS-PAGE on a PhastGel Gradient 4–15%, using the PhastSystem (GE Healthcare, Buckinghamshire, UK). The electrophoretic analysis was performed as described in [26 (link)].
The fractions eluted from Sepharose CL-6B were analyzed by SDS-PAGE on a PhastGel Gradient 4–15%, using the PhastSystem (GE Healthcare, Buckinghamshire, UK). The electrophoretic analysis was performed as described in [26 (link)].
5
Biofilm Extraction and Fractionation
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After separation from the planktonic culture, the biofilm formed at the air–liquid interface was suspended in 0.1 M phosphate buffer (pH 7.2) and sonicated twice (37 kHz, 40 °C, 30 min). Bacterial cells from the biofilm (280 mg) were pelleted by centrifugation (3000× g, 40 min), resuspended twice in acetone and dried in air at room temperature. The supernatant containing crude BM was dialysed against distilled water for 2 d, evaporated at 40 °C under reduced pressure (Laborota 4000; Heidolph, Schwabach, Germany) and lyophilised in a Benchtop 2K freeze dryer (VirTis, Gardiner, NY, USA).
The crude BM (20 mg) was redissolved in water and fractionated by gel permeation chromatography (GPC) on a Sepharose CL-6B column (2.5 × 46 cm; GE Healthcare, Chicago, IL, USA), by using 0.025 M NH4HCO3 (pH 8.3) as the eluent, and monitoring with a differential refractometer (2142; LKB, Bromma, Sweden). Both fractions, BM1 (10 mg) and BM2 (4 mg), were further assayed for biopolymer composition.
The polysaccharide fraction (BM3) was obtained by a degradation of the crude BM (100 mg) with aqueous 2% acetic acid (100 °C, 4 h), followed by GPC on a column of Sephadex G 50 (S) (56 × 2.6 cm, GE Healthcare, Chicago, IL, USA), using 0.05 M pyridinium acetate (pH 4.5) as the eluent, and monitoring with a differential refractometer (2142; LKB, Bromma, Sweden). The yield of BM3 was 15% of the crude BM.
The crude BM (20 mg) was redissolved in water and fractionated by gel permeation chromatography (GPC) on a Sepharose CL-6B column (2.5 × 46 cm; GE Healthcare, Chicago, IL, USA), by using 0.025 M NH4HCO3 (pH 8.3) as the eluent, and monitoring with a differential refractometer (2142; LKB, Bromma, Sweden). Both fractions, BM1 (10 mg) and BM2 (4 mg), were further assayed for biopolymer composition.
The polysaccharide fraction (BM3) was obtained by a degradation of the crude BM (100 mg) with aqueous 2% acetic acid (100 °C, 4 h), followed by GPC on a column of Sephadex G 50 (S) (56 × 2.6 cm, GE Healthcare, Chicago, IL, USA), using 0.05 M pyridinium acetate (pH 4.5) as the eluent, and monitoring with a differential refractometer (2142; LKB, Bromma, Sweden). The yield of BM3 was 15% of the crude BM.
6
Purification of Lactobacillus Plantarum EPS
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Lactobacillus plantarum YW11 was cultured in 10 L of pasteurized (85°C, 5 min) skim milk medium at 37°C for 18 h. The EPS produced by the strain was then isolated and prepared using the method previously reported (Wang et al., 2015a) . Briefly, after fermentation, 80% (wt/vol) trichloroacetic acid (TCA) was added to achieve the 4% (wt/vol) trichloroacetic acid concentration. The mixture was stirred at room temperature for 2 h, and then centrifuged at 12,000 × g for 30 min at 4°C. The cold ethanol (4°C) was added to the supernatant to achieve 70% concentration and then kept at 4°C overnight. The pellet of crude EPS was collected after centrifugation at 12,000 × g for 30 min at 4°C. For purification, the EPS was dissolved in distilled H 2 O (20 mg/mL) and fractionated with a diethylaminoethyl cellulose column. Peak fractions containing EPS were lyophilized and fractionated by Sepharose CL-6B column (GE Healthcare, Pittsburgh, PA). The fraction of pure EPS was lyophilized into powder and stored at -20°C for further use.
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