mAbs specific for the ectodomains of human receptors [αLOX-1 (15C4) (Li et al., 2012 (link)), αDC-ASGPR (49C11) (Li et al., 2012 (link)), αDCIR (9E8) (Klechevsky et al., 2010 (link)), αCD40 (12E12) (Flamar et al., 2013 (link)), αDectin-1 (15E2) (Ni et al., 2010 (link)), αDEC205 (MG38) (Bonifaz et al., 2002 (link)), and αLangerin (4C7)] were used. mAbs specific for the ectodomains of human MARCO (11A8), CLEC6 (9B9), and DC-SIGN/L (16E7) were generated using receptor ectodomain.hIgG (human IgG1 Fc) and human placental alkaline phosphatase (AP), as previously described (Ni et al., 2010 (link)). Cloned mAbs were purified by HPLC using MabSelect resin (GE Healthcare). The specificities of mAbs were verified by their specific binding to corresponding receptors expressed on 293F cells transfected with the full-length receptors. The specificities of the mAbs were also confirmed by ELISA by comparing them to the recombinant receptor-Fc and hIgG-Fc fusion proteins (Ni et al., 2010 (link)). Chimeric mAbs containing human IgG4 heavy chain with two site mutations (S228P and L235E) (Reddy et al., 2000 (link)) were made to further abolish non-specific binding to Fc receptors.
Mabselect resin
Manufactured by GE Healthcare
MabSelect resin is a protein A affinity chromatography medium designed for the capture and purification of monoclonal antibodies. The resin consists of a cross-linked agarose matrix with immobilized recombinant protein A ligands. MabSelect resin offers high binding capacity, recovery, and purity for efficient monoclonal antibody purification.
Automatically generated - may contain errors
Lab products found in correlation
Bira enzyme, by BPS Biosciences (3 mentions)
Amicon 10 kda spin concentrators, by Merck Group (3 mentions)
Nickel nta spin columns, by Qiagen (3 mentions)
Expi293 expression system, by Thermo Fisher Scientific (2 mentions)
Superdex 200 10 300 column, by GE Healthcare (1 mentions)
Pierce igg elution buffer, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)
Dynamis medium, by Thermo Fisher Scientific (1 mentions)
Fectopro reagent, by Polyplus Transfection (1 mentions)
Expicho expression system, by Thermo Fisher Scientific (1 mentions)
8 protocols using mabselect resin
1
Generation and Characterization of Monoclonal Antibodies Targeting Human Receptors
2
Protein Purification and Characterization
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Cell culture samples were purified by affinity chromatography using MabSelect resin (GE Healthcare). The purified samples were washed with 5 mM succinic acid, pH 5.8 and eluted with a 25 mM glycine, 25 mM succinic acid, pH 3.0 buffer. The samples were neutralized to pH 6–8 with 2 M Tris. The purified samples were used to analyze protein purity, charge variants, size variants, and N‐linked glycosylation.
3
SARS-CoV-2 S-RBD and hACE2-ECD Protein Production
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Reference SARS-CoV-2 S-RBD (nt 22,517 – 23,185; MN908947) and hACE2-ECD (aa19 – aa617; Uniprot Q9BYF1) coding gene fragments were obtained by chemical synthesis. S-RBD reference gene with C-terminus hexahistidine tag was cloned in 5’NotI/3’BamHI restriction sites of pSCSTa plasmid under the control of CMV promoter and used to generate mutant constructs (G476S, V483A, H519Q, and A520S) by oligonucleotide-mediated PCR mutagenesis. All S-RBD proteins were transiently produced in FreeStyle 293f cells (Invitrogen). Culture supernatant containing protein was bound to Ni-NTA resin (Yeason Biotech), eluted with 500 mM imidazole in 20 mM HEPES/500 mM NaCl, buffer exchanged to 1x PBS and further cleaned by size exclusion chromatography using Superdex 200 10/300 column (GE Healthcare) on AKTA Avant150 FPLC system. The Human ACE2 gene was cloned into unique SfiI restriction sites in pFUSE-mIgG2A-Fc2 plasmid (Invivogen) under the control of a hEF1-HTLV-1 promoter and produced like S-RBD proteins. Culture supernatant containing hACE2-ECD-mFc was bound to Mabselect resin (GE Healthcare), eluted by Pierce IgG elution buffer (Thermo Scientific), and buffer exchanged to 1x PBS. All recombinant proteins were resolved on 4 – 12% gradient SDS-PAGE to ascertain purity and correct size.
4
Expression and Purification of IL-2 Fusion Proteins
5
Fab and IgG Protein Expression
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EPHA2-specific Fab fragments selected from antibody libraries were re-cloned into the pET22a plasmid and expressed in the periplasm of BLgold. After growing bacteria to OD600 = 0.5, Fab expression was induced by addition of 1 mmol/L IPTG and incubation of culture at 28°C for 15 hours. Fabs were expressed with heavy chain C-terminal c-Myc and His6 tags (CH-AAA-c-Myc-GAALE-His6) for assisting purification and detection. Fab fragments were purified using Ni+-resin (Profinity IMAC Resin, Ni-charged, Bio-Rad, 1560135).
IgGs were expressed in CHO-S culture grown in Dynamis medium (Thermo Fisher Scientific, A2661501) following transfection with the FectoPro reagent (Polyplus Transfection, 116–010). The antibodies were harvested on days 12–14 after transfection and purified using MabSelect resin (GE Healthcare, 17–5199–01). After binding in PBS, the antibodies were eluted with 100 mmol/L citrate buffer (pH 3.0) followed by immediate titration to pH = 5.5 using 1 mol/L Tris-HCl, pH = 8.0. The eluted protein was dialyzed against and stored in the formulation buffer composed of 20 mmol/Ll -Histidine, 30 mmol/L Citric acid, 32 mmol/L Na2HPO4, pH = 6.0, 1% trehalose, 0,05% Tween-20.
IgGs were expressed in CHO-S culture grown in Dynamis medium (Thermo Fisher Scientific, A2661501) following transfection with the FectoPro reagent (Polyplus Transfection, 116–010). The antibodies were harvested on days 12–14 after transfection and purified using MabSelect resin (GE Healthcare, 17–5199–01). After binding in PBS, the antibodies were eluted with 100 mmol/L citrate buffer (pH 3.0) followed by immediate titration to pH = 5.5 using 1 mol/L Tris-HCl, pH = 8.0. The eluted protein was dialyzed against and stored in the formulation buffer composed of 20 mmol/L
6
Production and Purification of Fc-modified Nic•mAbs
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To reduce/eliminate Fc effector function (complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity), which is not needed for binding and neutralizing nicotine and could be a liability in the event of cross-reactivity with membrane targets, 5G4, 7A8, 12F5, and 8D1 were reformatted from the IgG1 to the IgG4 isotype [40 (link)] and named ATI-1010 through ATI-1013, respectively. Additional Fc sequence modifications were concurrently made to prevent Fab-arm exchange in vivo [41 (link)]. The fully human nic•mAbs ATI-1010, 1011, 1012, and 1013 were produced in sufficient quantity for in vivo testing using the ExpiCHO Expression System (Thermo-Fisher). Essentially, ExpiCHO cells were expanded at 37°C in 2L shake flasks. Cultures were adjusted to 6x106 cells/mL and transfected per the vendor-supplied protocol. Cultures were fed on days 1 and 5 post-transfection, and the temperature was lowered to 32°C on day 1. Cultures were harvested on day 12 by centrifugation, 0.22 μm filtered, and stored at 4°C. Purification was performed on a Protein A affinity column (MAbSelect resin (GE Life Sciences)) with glycine pH 3.0 elution. After neutralization and pooling of the main peak fractions, the material was exchanged into PBS by dialysis. Purity was ≥95% by SDS-PAGE with Coomassie blue staining.
7
Expression and Purification of IL-2 Fusion Proteins
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IL-2/Fc fusion proteins were expressed using the Expi293 expression system according to manufacturer instructions (Thermo Scientific). Proteins were constructed as human IgG1 Fc fusions at the N- or C terminus to human IL-2 through a (G4S)4 linker. C-terminal fusions omitted the C-terminal lysine residue of human IgG1. The AviTag sequence GLNDIFEAQKIEWHE was included on whichever terminus did not contain IL-2. Fc mutations to prevent dimerization were introduced into the Fc sequence (Ishino et al., 2013 (link)). Proteins were purified using MabSelect resin (GE Healthcare). Proteins were biotinylated using BirA enzyme (BPS Biosciences) according to manufacturer instructions, and extensively buffer-exchanged into phosphate buffered saline (PBS) using Amicon 10 kDa spin concentrators (EMD Millipore). The sequence of IL-2Rβ/g Fc heterodimer was based on a reported active heterodimeric molecule (patent application US20150218260A1), with the addition of (G4S)2 linker between the Fc and each receptor ectodomain. The protein was expressed in the Expi293 system and purified on MabSelect resin as above. IL2-Rα ectodomain was produced with C-terminal 6xHis tag and purified on Nickel-NTA spin columns (QIAGEN) according to manufacturer instructions.
8
Purification and Characterization of IL-2/Fc Fusion Proteins
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The Expi293 expression system was used to express IL-2/Fc fusion proteins. Expression was conducted as prescribed by the manufacturer instructions (Thermo Scientific). Proteins were formulated as the Fc of human IgG1 fused at its N-or C-terminus to human IL-2 using a (G4S)4 linker. C-terminal lysine residues of human IgG1 were not included in C-terminal fusions. The AviTag sequence GLNDIFEAQKIEWHE was added to the Fc terminus which did not contain IL-2. Fc mutations which prevented dimerization were introduced into the Fc sequence for monovalent muteins 61 . MabSelect resin (GE Healthcare) was used to purify protein. Biotinylation of proteins was conducted using BirA enzyme (BPS Biosciences) according to manufacturer instructions. Extensive buffer-exchanging into phosphate buffered saline (PBS) was conducted using Amicon 10 kDa spin concentrators (EMD Millipore). The sequence which was used to express the IL2Rβ/γ Fc heterodimer was the same as that of a reported, active heterodimeric molecule (patent application US20150218260A1); a (G4S)2 linker was added between the Fc portion and each receptor ectodomain. The Expi293 system was used to express the protein, which was subsequently purified on MabSelect resin as above. The IL2Rα ectodomain was generated to include a C-terminal 6xHis tag and then purified on Nickel-NTA spin columns (Qiagen) according to manufacturer instructions.
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