For conjugation of targeting ligands (both antibodies and SOD), NPs were prepared and incubated overnight with either antibodies or radiolabeled SOD (as above) at 4°C. Immuno-NPs (~50mAbs/liposome) were purified using a 20mL Sepharose 4B-Cl column (GE).
Sepharose cl 4b column
Manufactured by GE Healthcare
Sourced in United States
Sepharose CL-4B column is a size exclusion chromatography medium used for the separation and purification of biomolecules based on their size and molecular weight. It is composed of cross-linked agarose beads that provide a porous structure for the separation process. The column can be used to fractionate a variety of molecules, including proteins, nucleic acids, and other macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Kta start system, by GE Healthcare (2 mentions)
Alexa fluor 790, by Thermo Fisher Scientific (1 mentions)
Odyssey m infrared imager, by LI COR (1 mentions)
Peristaltic pump, by GE Healthcare (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Promega (1 mentions)
Proteinase k, by Roche (1 mentions)
Protease inhibitor, by Roche (1 mentions)
8 protocols using sepharose cl 4b column
1
Antibody-Conjugated Nanoparticle Purification
2
Antibody-Conjugated Nanoparticle Purification
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For conjugation of targeting ligands (both antibodies and SOD), NPs were prepared and incubated overnight with either antibodies or radiolabeled SOD (as above) at 4°C. Immuno-NPs (~50mAbs/liposome) were purified using a 20mL Sepharose 4B-Cl column (GE).
3
Antibody-Conjugated Liposome Preparation
4
In Vivo Tracking of Extracellular Vesicles
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For in vivo tracking studies, CPC-EVs were labeled with Alexa Fluor 790- or Alexa Fluor 647 NHS ester dyes (Thermo Fisher Scientific). EVs were incubated with 0.05 nM reactive dye in 0.1 M NaHCO3 in phosphate-buffered saline (PBS) and incubated for 30 min at 37°C while shaking at 450 rpm. After labeling, the free amine-reactive dye was quenched using a final concentration of 0.1 M Tris-HCl for 30 min at RT. Quenched free dye was removed using a Sepharose CL-4B column coupled to an ÄKTA start system (GE Healthcare) containing a UV 280 nm flow cell. EV-containing fractions were concentrated using a 100 kDa MWCO Amicon Ultra-4 spin filter (Merck Millipore). Fluorescent labeling efficiency was determined by diluting different EV volumes in 50 µl PBS and measuring fluorescence at 800 nm using an Odyssey M Infrared Imager (LI-COR Biosciences).
5
Purification and Characterization of T. cruzi Antigens
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The total shed material was 2-fold diluted with 200 mM ammonium acetate (pH 6.5) and loaded onto a Sepharose CL-4B column (1 × 40 cm, GE Healthcare, Piscataway, NJ) preequilibrated with 100 mM ammonium acetate (pH 6.5). The column was eluted with the equilibration buffer, in a flow rate of 0.2 mL/min using a peristaltic pump (GE Healthcare). Fractions (N = 80) of 1 mL were collected and then screened by chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) as described elsewhere [20 (link)], using anti-T. cruzi membrane polyclonal antibody (mouse) or anti-Alpha Gal purified from sera of chronic Chagasic patients (human Ch anti-αGal), as described [21 (link)]. The most reactive fractions being pooled and concentrated in a vacuum centrifuge and then resuspended in filtered PBS for further analysis by nanoparticle tracking analysis (NTA) as previously described [20 (link), 29 , 30 (link)].
6
HCGβ 81–95 Peptide-functionalized Nanoparticles
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Peptide HCGβ 81–95 (SYAVALSCQCALCRR) and peptide FITC-labeled HCGβ 81–95 (FITC-SYAVALSCQCALCRR) were synthesized and purified by high-performance liquid chromatography (HPLC). Nanoparticles were prepared with maleimide-PEG-PLA, which were synthesized by a ring-opening polymerization of lactide initiated by the hydroxyl group of methoxy poly (ethylene glycol), using the emulsion/solvent evaporation technique.23 (link),24 (link)
MTX-NP was prepared by an oil-water emulsification solvent evaporation method. MTX (10 mg) was dissolved in 2 mL of dichloromethane containing 2.5% maleimide-PEG-PLA and MPEG-PLA. Add organic phase into 20 mL of 1% sodium cholate aqueous solution and sonicated. Then, stir the emulsion for 1 h at 40°C to obtain MTX-NPs (5 mg/mL).
To prepare HCG81-NP, the mixture of HCGβ 81–95 peptide and nanoparticles (the molar ratio of HCGβ 81–95 peptide and maleimide was 1:1) was magnetically stirred overnight. The reaction mixture was then isolated on a Sepharose CL-4B column (GE Healthcare) by elution with HEPES buffer. The milky HCG81-NP fractions were collected by centrifugation. The working model of HCG81-NP was shown inSupplementary Figure S4
MTX-NP was prepared by an oil-water emulsification solvent evaporation method. MTX (10 mg) was dissolved in 2 mL of dichloromethane containing 2.5% maleimide-PEG-PLA and MPEG-PLA. Add organic phase into 20 mL of 1% sodium cholate aqueous solution and sonicated. Then, stir the emulsion for 1 h at 40°C to obtain MTX-NPs (5 mg/mL).
To prepare HCG81-NP, the mixture of HCGβ 81–95 peptide and nanoparticles (the molar ratio of HCGβ 81–95 peptide and maleimide was 1:1) was magnetically stirred overnight. The reaction mixture was then isolated on a Sepharose CL-4B column (GE Healthcare) by elution with HEPES buffer. The milky HCG81-NP fractions were collected by centrifugation. The working model of HCG81-NP was shown in
7
Adenovirus Particle Production Protocol
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Adenovirus particles were produced following the manufacturer’s protocol (AdEasy™ XL Adenoviral Vector System, Stratagene, USA). The recombinant adenovirus plasmids were transfected into AD-293 cells by using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA). All the adenoviruses generated were purified by CsCl gradient ultracentrifuge and desalted using a Sepharose CL-4B column (GE Healthcare, Waukesha, WI, USA), and the viral concentrations were determined.
8
Proteinase K-based EV Purification
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EVs were incubated in a final concentration of 100 µg/mL Proteinase K (Promega) for 30 min at 37 °C. Proteinase K was inactivated by diluting the EV sample to 1 mL in PBS supplemented with protease inhibitor (Roche), and EVs were subsequently separated from fractionated proteins by SEC using a Sepharose CL-4B column connected to an ÄKTA start system (GE Healthcare) containing an UV 280 nm flow cell. An EV sample without treatment with Proteinase K, but with the subsequent isolation and concentrating steps, was taken along in parallel and served as untreated control. EV-containing fractions were pooled and again concentrated using a 100-kDa MWCO Amicon Ultra-4 spin filter (Merck Millipore).
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