Purified CCF protein and GEM particles were prepared and stored according to previous protocols (19 (link), 20 (link)). Briefly, the CCF protein was expressed by Escherichia coli Rosetta (DE3) cells with pET-28a-CCF. The protein was first purified by nickel affinity chromatography (GE Healthcare), followed by anion-exchange chromatography with DEAE Sepharose FF (Amersham Pharmacia Biotech AB, Sweden). The purity of CCF was confirmed by Coomassie blue staining. The GEM particles were prepared by Lactococcus lactis NZ9000 cells using a hot-acid water bath. Vaccine with Alum was prepared with an equal volume of CCF solution and Alum adjuvant. CpG ODN 1826 was obtained from Sangon Biotech Co., Led. (China, Shanghai) and dissolved in CCF solution before intranasal vaccination.
Deae sepharose ff
Manufactured by Cytiva
Sourced in Sweden
DEAE Sepharose FF is a strong anion exchange resin used for the purification and separation of biomolecules. It is composed of cross-linked agarose beads with diethylaminoethyl (DEAE) functional groups. The resin is designed for high-performance liquid chromatography (HPLC) applications, providing efficient separation and recovery of target molecules.
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Lab products found in correlation
4 protocols using deae sepharose ff
1
Purification and Preparation of CCF Protein and GEM Particles
2
Purified Multivalent Vaccine Formulations
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Purified CTB-UE-CF (CCF) protein was constructed by cholera toxin B, multi-epitopes from H. pylori urease, and self-adjuvant regions from S typhimurium phase I flagellin FliC, and the preparation of CCF was undertaken as described previously [19 (link)]. Briefly, CCF protein was expressed by Escherichia coli Rosetta (DE3) cells with pET-28a-CCF. First, the protein was purified using nickel-affinity chromatography (GE Healthcare, Amersham, UK), followed by anion-exchange chromatography with DEAE Sepharose FF (Amersham Pharmacia Biotech Uppsala, Sweden). Alum-based vaccine (Al+CCF) was prepared with an equal volume of CCF solution and alum adjuvant. A silk fibroin hydrogel-based vaccine (SF+CCF) was prepared: CCF solution was mixed with silk fibroin protein, and then mixed with an equal volume of 90% polyethylene glycol-400. The final concentration of CCF and silk fibroin protein was 1.5 and 40 mg/mL, respectively. Per mouse was injected with 10.5 μg/mouse.
3
Purification of Antibacterial Proteins from Algae
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For each sample, 274 g of algal fronds were homogenized with 25 mM Tris-HCl (pH 7.2) and centrifuged at 15,000 g for 15 min. Ammonium sulfate was added to the supernatant to yield a 100% saturated solution and proteins were precipitated by centrifugation. The pellets were resuspended in 25 mM Tris-HCl buffer (pH 7.2) and dialyzed overnight against 25 mM Tris-HCl buffer (pH 7.2) to remove salt. The dialyzed samples were subjected to anion exchange chromatography on DEAE Sepharose FF (250 ml, Amersham Biosciences) and the column was eluted with 0.17 M, 0.27 M, and 0.37 M NaCl in Tris-HCl buffer (pH 7.2). The 0.27 M NaCl fraction, which had antibacterial activity, was subjected to gel filtration chromatography using Toyopearl HW-65S resin (particle size 20–40 μm, Tosoh), and the column was eluted with 25 mM Tris-HCl buffer (pH 7.2) at a 24 ml/min flow rate.
4
Purification and Formulation of CCF Vaccine
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The preparation and storage of purified CCF protein, a dual-antigen epitope and dual-adjuvant vaccine constructed by cholera toxin B, multiepitopes from H. pylori urease, and self-adjuvant regions from S. typhimurium phase I flagellin FliC, called CTB-UE-CF (CCF), were performed in previous protocols [15 (link)]. Briefly, the CCF protein was expressed by Escherichia coli Rosetta (DE3) cells with pET-28a-CCF. The protein was first purified using nickel affinity chromatography (GE Healthcare), followed by anion-exchange chromatography with DEAE Sepharose FF [16 (link)] (Amersham Pharmacia Biotech AB, Sweden). The purity of CCF was confirmed using the Coomassie blue staining. Vaccine with alum was prepared with equal volumes of CCF solution and alum adjuvant.
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