Nonlipidated recombinant fHBP (rP2086) variants were expressed and purified as previously described (25 (link)). Mutations in the MN86-994-11 binding epitope were introduced by site-directed mutagenesis. In this case, a His-tagged version of rP2086-B01 cloned into plasmid vector pET30a was used as the mutagenesis template to facilitate the purification of recombinant mutants. A mutagenesis kit was used in accordance with the manufacturer’s instructions (QuikChange; Agilent), mutagenic oligonucleotides used in the reaction were designed with the QuikChange Primer Design Program, and the presence of intended mutations and the absence of secondary mutations were confirmed by DNA sequencing. Mutant proteins expressed in Escherichia coli BL21(DE3) were purified by Ni Sepharose affinity chromatography and size exclusion chromatography (GE Healthcare). All CD and ITC experiments were done with 1× PBS, pH 7.4. Protein and antibody samples were thoroughly dialyzed against experimental buffer. Concentrations of rP2086-B01 and MN86-994-11 were determined spectrophotometrically by using extinction coefficients of 0.363 and 1.4 (mg/ml)−1 cm−1 at 280 nm, respectively. Light scattering was taken into account as previously described (42 (link)).
Ni sepharose affinity chromatography
Manufactured by GE Healthcare
Ni Sepharose is an affinity chromatography resin used for the purification of histidine-tagged proteins. It consists of Sepharose beads to which nickel ions (Ni2+) are chelated. The Ni2+ ions bind to the histidine tags on recombinant proteins, allowing them to be selectively captured and purified from complex samples.
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Lab products found in correlation
Size exclusion chromatography, by GE Healthcare (1 mentions)
Quikchange, by Agilent Technologies (1 mentions)
Hitrap q column, by GE Healthcare (1 mentions)
Superdex 200 size exclusion column, by GE Healthcare (1 mentions)
Akta pure system, by Cytiva (1 mentions)
Protein a affinity chromatography, by GE Healthcare (1 mentions)
Protein l affinity chromatography, by GE Healthcare (1 mentions)
Sars cov 2 s1, by Sino Biological (1 mentions)
Sars cov 2 rbd, by GenScript (1 mentions)
Anti his tag antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using ni sepharose affinity chromatography
1
Recombinant fHBP Variant Purification and Characterization
2
Purification of Recombinant Hsp90β Variants
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Escherichia coli BL21 (DE3) cells were transformed with pET-15b-HSP90β plasmids expressing the wild-type or mutant forms (C366A, C412A, C562A and C589A) of Hsp90β and cultured at 37 °C. Protein production was induced by 0.5 mM isopropyl β-d -thiogalactopyranoside (IPTG) for 16 h. Recombinant proteins were then affinity purified by using Ni Sepharose affinity chromatography (GE Healthcare). Subsequently, His-Hsp90β was purified by ion-exchange chromatography with a HiTrap Q column (GE Healthcare). The protien was eluted at 380 mM NaCl, 50 mM Tris-HCl, pH8.0. His-Hsp90β was further separated by a Superdex 200 size exclusion column (GE Healthcare). The fractions containing His-Hsp90β were collected and pooled. The purified proteins were stored in 50 mM Tris-HCl, 380 mM NaCl, 1 mM DTT, pH8.0.
3
Purification of Fusion Protein for Immunoassay
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The purification was performed through a Ni-Sepharose affinity chromatography (GE Healthcare Life Sciences). Briefly, the resin was washed and eluted by gradually increasing imidazole concentrations. The fractions were analyzed on a 15% SDS-PAGE and concentrated in 10 kDa centrifuge devices (Amicon, Miami, FL). Subsequently, another purification step was performed by size exclusion chromatography on a Superdex G75 10/300 column (GE Healthcare Life Sciences) using the AKTA-pure system (Cytiva, Marlborough, MA). The samples were evaluated by Dynamic Light Scattering (DLS) in a Zetasizer Nano series S90 device (Malvern Instruments, Malvern, UK), and data acquisition was performed from 10 cycles of the 30 s at a constant temperature of 25 °C. The purified protein was also tested in an immunoenzymatic assay, using serum samples from goats and sheep naturally infected or not by C. pseudotuberculosis in different dilutions, and the MBP:PLD:CP40 purified protein as antigen and in different concentrations, with the objective to verify the recognition of the fusion protein by antibodies of animals with caseous lymphadenitis.
4
Bispecific Antibody Production and Purification
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A simplified expression and purification schematic was developed in Fig. 3a . Briefly, Antibody fragments were purified by protein L affinity chromatography (GE Healthcare) from filtered cell culture supernatants referring to standard protocols. The supernatants were loaded and equilibrated with binding buffer (20 mM sodium phosphate, 500 mM NaCl, pH 7.4). Elution of antibodies was achieved at pH 2.8 (0.1 M sodium citrate) with a 20 CV linear gradient from 0% to 100%, followed by immediate neutralization of the sample. The samples were dialyzed overnight to splicing buffer (10 mM Tris-HCl, 0.5 M NaCl, pH 7.9), followed by splicing reaction of the antibody fragments catalyzed by the split intein. Imidazole were added into the splicing product at 30 mM final concentration, then the reaction mixture went into the Ni Sepharose affinity chromatography (GE Healthcare) and flowthrough containing the BsAb was collected. The flowthrough collect was further purified through protein A affinity chromatography (GE Healthcare). Antibody was eluted at pH 2.8 (0.1 M sodium citrate) with an elution of 20 CV linear gradient from 0% to 100%. The eluates were neutralized immediately, dialyzed overnight to PBS, and filter-sterilized over 0.22 μM dead-end filters. The protein concentration of the samples was measured by BCA protein assay kit.
5
SARS-CoV-2 Antibody Binding Assay
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All recombinant proteins used in this study were His-tagged for normalization. SARS-CoV-2 and SARS-CoV N proteins were purified from pcDNA3.1-SARS-CoV-2 N or pDual-GC-SARS-CoV N transfected HEK293 T cells using Ni Sepharose affinity chromatography (GE Healthcare). SARS-CoV-2 S1 and SARS-CoV S1 were obtained from Sino Biological. SARS-CoV-2 RBD and SARS-CoV RBD were custom produced by GenScript. The amino acid sequence of these recombinant proteins are given in Supplementary Table 2. 25 μg of each antigen was coupled onto MagPlex-c microsphere (Luminex) using xMAP antibody coupling kit (Luminex) according to the manufacturer’s instructions. To assess the antibody reactivity, 1250 beads/antigen were incubated with various serum samples at 1:100 dilution in PBS containing 1% BSA for an hour at 37°C with agitation. The bound antibodies were detected with goat anti-human IgG, PE (eBioscience) at 1:1000 dilution. The level of antibody binding was determined using the Luminex MAGPIX system. The readings were normalised by dividing with the readings obtained from the anti-His tag antibody (Thermo Scientific) and presented as net mean fluorescence intensity (MFI).
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