The recombinant JEV P3 strain EDIII fragments were expressed and purified as previously described (Du et al., 2013 (link)). Briefly, JEV EDIII fragments (residues 292-402; accession number AY243844) were expressed in HEK293T cells and secreted into the cell culture medium. EDIII, EDIII-EDIII, EDIII-Fd and E with a C-terminal His6 tag were purified on Ni-NTA column (Qiagen), and EDIII-Fc was purified on Protein A affinity column (GE Healthcare). The yeild of used expression and purification system for each recombinant protein is larger than 0.5 mg/L, and the purity of each recombinant protein is higher than 95%.
Protein a affinity column
Manufactured by GE Healthcare
Sourced in United States
The Protein A affinity column is a piece of laboratory equipment used for the purification and isolation of antibodies from complex mixtures. It utilizes the high-affinity interaction between Protein A, a bacterial protein, and the Fc region of immunoglobulins to selectively capture and concentrate antibodies. The column allows for the efficient separation and recovery of antibodies from samples such as cell culture supernatants, ascites fluids, or other biological sources.
Automatically generated - may contain errors
Lab products found in correlation
Pfuse chig hg1, by InvivoGen (2 mentions)
Pfuse2 clig hk, by InvivoGen (2 mentions)
Superdextm 200 increase 10 300 gl column, by GE Healthcare (2 mentions)
Ni nta column, by Qiagen (1 mentions)
Pfuse chig hg1, by Thermo Fisher Scientific (1 mentions)
Psectag2 a, by Thermo Fisher Scientific (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Superdextm 200 10 300 gl, by GE Healthcare (1 mentions)
Elx800 automatic microplate reader, by Agilent Technologies (1 mentions)
Histrap excel column, by GE Healthcare (1 mentions)
Superdextm 200 10 300 gl column, by GE Healthcare (1 mentions)
Cho s cells, by Thermo Fisher Scientific (1 mentions)
Turbocapture 96 mrna kit, by Qiagen (1 mentions)
Pierce fab preparation kit, by Thermo Fisher Scientific (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Maxisorp immunoplate, by Thermo Fisher Scientific (1 mentions)
Immobilized pepsin, by Thermo Fisher Scientific (1 mentions)
Protein g chromatography, by GE Healthcare (1 mentions)
Histrap hp affinity column, by GE Healthcare (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
17 protocols using protein a affinity column
1
JEV EDIII Protein Production and Purification
2
Generating Anti-GPC3 Antibody Constructs
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The sequence of full-length GPC3 was cloned into the pLVX vector. For protein purification, truncated GPC3 (Q25-S550) was fused to a human Fc tag and cloned into the pFUSE vector. The 32A9 or 42A1 scFv sequence, isolated from the Tomlinson I library to bind human GPC3 [25 (link), 26 (link)], was fused to the human Fc tag and cloned into the pFUSE vector. The 32A9 or 42A1 heavy chain variable region and light chain variable region sequences were amplified by adding the IL-2 signal peptide and were inserted into the expression vectors pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk (Invitrogen, San Diego, CA), respectively. All pFUSE plasmids were identified by sequencing and then transfected into 293 T cells. After collecting the supernatant, protein purification was performed using a protein A affinity column (GE Healthcare, Milwaukee, WI, USA). GPC3-His and GPC5-His proteins were purchased from R&D (Minneapolis, MN, USA).
YP7, which is a control mouse monoclonal antibody against GPC3 [27 ], was used to evaluate GPC3 expression by immunohistochemistry staining, flow cytometry and ELISA.
YP7, which is a control mouse monoclonal antibody against GPC3 [27 ], was used to evaluate GPC3 expression by immunohistochemistry staining, flow cytometry and ELISA.
3
Cloning and Purification of GPC3 and Antibodies
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The sequence of full-length GPC3 was cloned into the pLVX vector. For protein purification, truncated GPC3 (Q25-S550) was fused to a human Fc tag and cloned into the pFUSE vector. All point mutants of GPC3 and the GPC3 mutant lacking the HS chains (GPC3ΔHS) were generated by introducing point mutations using the overlapping PCR method. The mFrizzled8 ECD-hFc plasmid was purchased from Addgene (Cambridge, MA). The 32A9 scFv sequence was fused to the human Fc tag and cloned into the pFUSE vector as well. The 32A9 heavy chain variable region and light chain variable region sequences were amplified by adding IL-2 signal peptide and were inserted into the expression vectors pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk (Invivogen, San Diego, CA), respectively. All plasmids were identified by sequencing and then transfected into 293T cells. After collecting the supernatant, protein purification was accomplished with a protein A affinity column (GE Healthcare, Milwaukee, WI). GPC3-his and GPC5-his proteins were purchased from R&D (Minneapolis, MN).
YP7, a control mouse monoclonal antibody against GPC3 [28 ], was used to evaluate GPC3 expression in immunohistochemistry staining, flow cytometry and ELISA assays.
YP7, a control mouse monoclonal antibody against GPC3 [28 ], was used to evaluate GPC3 expression in immunohistochemistry staining, flow cytometry and ELISA assays.
4
Purification of BAFF-Trap Protein
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First, CHO cells were transfected with the recombinant plasmid, pEF1/V5-BAFF-Trap. Second, cloned strains that stably and efficiently expressed BAFF-Trap were obtained through G418 (2 μg/ml) (Sigma-aldrich, Saint Louis, USA) selection. After intermediate culture of BAFF-Trap-expressing cells, the culture supernatant was collected and loaded onto a protein-A affinity column (GE, Pittsburgh, USA) equilibrated with binding buffer (20 mm phosphate buffer, pH 7.2). The column was washed with binding buffer and further washed with 0.1 m citrate sodium buffer (pH 3.6). Then the pH of the solution harboring BAFF-Trap was adjusted to 7.2 with Tris buffer. After dialysis with 20 mm PB (pH 7.2), the protein solution was loaded onto a SP chelating Sepharose column (GE, Pittsburgh, USA) equilibrated with 20 mm PB (pH 7.2). The column was washed with 20 mm (pH 7.2) and the hybrid protein was detached with 20 mm PB (pH 7.2) and 100 mm NaCl buffer. The target protein was eluted with 20 mm PB (pH 7.2) and 300 mm NaCl buffer. Eventually, the protein solution was dialyzed with phosphate-buffered saline (PBS).
5
Recombinant ErbB2 Extracellular Domain Production
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The extracellular domain (ECD) of ErbB2 in fusion to an enterokinase cleavage peptide and a human IgG1 Fc fragment was subcloned into the mammalian expression vector pSectag 2A (Invitrogen). The recombinant dimeric ErbB2 ECD-Fc fusion protein was transiently expressed in Expi293 cells using the Expifectamine reagent (Invitrogen), purified on Protein A affinity column (GE Healthcare), and then biotinylated (denoted Bio-ECD) using the EZ-Link NHS-PEG4-Biotin labeling kit (Pierce). To prepare recombinant ErbB2 ECD, the fusion protein was incubated with bovine enterokinase (Novoprotein) and passed through Protein A to remove the cleaved Fc fragment.
6
Expression and Purification of hACE2-mFc and SARS-CoV-2 RBDs
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The hACE2 fused with mFc were expressed and purified from the culture supernatants of HEK293F cells using a Protein A affinity column (GE Healthcare) and further purified by gel filtration using a SuperdexTM 200 10/300 GL (GE Healthcare). Purified proteins were stored in a buffer containing 20 mM Tris-HCl and 150 mM NaCl (pH 8.0). Proteins for SPR assay were transferred to PBST (1.8 mM KH2PO4, 10 mM Na2HPO4 (pH 7.4), 137 mM NaCl, 2.7 mM KCl, and 0.005% (v/v) Tween 20) buffer.
The hACE2 and RBDs from original SARS-CoV-2 and VOCs cloned in pCAGGS were expressed in HEK293F cells. Cell culture supernatants were collected, filtered with a 0.22 μm filter, purified by His-Trap HP column (GE Healthcare), and SuperdexTM 200 Increase 10/300 GL column (GE Healthcare). Purified proteins were stored in protein buffer (20 mM Tris-HCl, pH 8.0 and 150 mM NaCl).
The hACE2 and RBDs from original SARS-CoV-2 and VOCs cloned in pCAGGS were expressed in HEK293F cells. Cell culture supernatants were collected, filtered with a 0.22 μm filter, purified by His-Trap HP column (GE Healthcare), and SuperdexTM 200 Increase 10/300 GL column (GE Healthcare). Purified proteins were stored in protein buffer (20 mM Tris-HCl, pH 8.0 and 150 mM NaCl).
7
Monoclonal Antibody Production and Purification
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For protein purification, the heavy chain variable region and light chain variable region sequences of parental antibodies (and their mutations) were amplified by adding IL-2 signal peptide and were inserted into expression vectors, pFUSE-CHIg-HG1 and pFUSE2-CLIg-hk (Invivogen, USA), respectively. 293T cells were transiently transfected with plasmids carrying the antibody heavy chain and the light chains at a 1:1 ratio.
After transfection, the supernatant was harvested daily for five consecutive days. Supernatant collected was then pooled and clarified by centrifugation (3000 g for 5 minutes, 4 °C) followed by filtration through a 0.45 μm filter. Affinity chromatography was used to purify expressed monoclonal antibodies using a Protein A affinity column (GE Healthcare, USA) able to bind to the Fc fragment. Purified antibodies were buffer-exchanged into phosphate buffer saline (PBS), concentrated using Amicon Ultra-4 10 kDa centrifugal filter units (Millipore Sigma, USA) and stored at 4 °C until use.
After transfection, the supernatant was harvested daily for five consecutive days. Supernatant collected was then pooled and clarified by centrifugation (3000 g for 5 minutes, 4 °C) followed by filtration through a 0.45 μm filter. Affinity chromatography was used to purify expressed monoclonal antibodies using a Protein A affinity column (GE Healthcare, USA) able to bind to the Fc fragment. Purified antibodies were buffer-exchanged into phosphate buffer saline (PBS), concentrated using Amicon Ultra-4 10 kDa centrifugal filter units (Millipore Sigma, USA) and stored at 4 °C until use.
8
Purification and Antibody Production of IgM from L. crocea
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L. crocea IgM purification and antibody production against the IgM were performed following the methods described for the production of a monoclonal antibody against smallmouth bass (Micropterus dolomieu) IgM (Ottinger et al. 2021 (link)). Serum IgM was purified by using a combination of affinity chromatography with a protein-A affinity column (GE Healthcare, Milwaukee, USA) and gel-filtration chromatography according to the previous method described by He et al. (2022 ). The purity and molecular weight of the purified IgM (LcIgMH) were examined by SDS-PAGE.
The purified LcIgMH was subsequently used as an antigen to inject into Kunming mice (SLAC, Shanghai, China) and produce murine anti-LcIgMH sera according to the previous description (Huang et al. 2013 (link); Ye et al. 2022 (link)). The antibody titer was detected by the enzyme-linked immunosorbent assay (ELISA) as described previously (Huang et al. 2002 (link), 2003 (link)). Briefly, purified LcIgMH was used as coating antigen, and murine anti-LcIgMH sera and horseradish peroxidase (HRP)–conjugated goat anti-murine immunoglobulin G (IgG) were used as the primary antibody and the secondary antibody, respectively. The optical density (OD) at 415 nm was read by ELx800 automatic microplate reader (BioTek, Vermont, USA). If the calculated absorbance value was > 0.1, the sample was considered positive.
The purified LcIgMH was subsequently used as an antigen to inject into Kunming mice (SLAC, Shanghai, China) and produce murine anti-LcIgMH sera according to the previous description (Huang et al. 2013 (link); Ye et al. 2022 (link)). The antibody titer was detected by the enzyme-linked immunosorbent assay (ELISA) as described previously (Huang et al. 2002 (link), 2003 (link)). Briefly, purified LcIgMH was used as coating antigen, and murine anti-LcIgMH sera and horseradish peroxidase (HRP)–conjugated goat anti-murine immunoglobulin G (IgG) were used as the primary antibody and the secondary antibody, respectively. The optical density (OD) at 415 nm was read by ELx800 automatic microplate reader (BioTek, Vermont, USA). If the calculated absorbance value was > 0.1, the sample was considered positive.
9
Purification of SARS-CoV-2 Proteins and Antibodies
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Protein A affinity column (GE Healthcare, USA) purification followed by gel filtration using a SuperdexTM 200 10/300GL column (GE Healthcare, USA) was used to purify the 14 ACE2 proteins fused with the mouse IgG Fc domain, 44 antibodies, and human ACE2 proteins fused with the human Fc tag from HEK293F cell culture supernatants. The purified proteins were then stored in phosphate-buffered saline (PBS) buffer. SARS-CoV-2 RBD, SARS-CoV-2 NTD, RsYN04 RBD, RsYN04-T484W RBD, and S43 nanobody with His tag were purified from the HEK293F cell culture supernatants using a His-Trap Excel column (GE Healthcare, USA), followed by purification using a SuperdexTM 200 Increase 10/300 GL column (GE Healthcare, USA). The purified proteins were then stored in buffer (20 mmol/L Tris-HCl, pH 8.0, and 150 mmol/L NaCl ).
10
Bispecific Antibody Production and Purification
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