Two blocking antibodies were used. CLS026 is a mouse IgG1 antibody specific for a neoepitope on murine C5a. CLS026 was derived using a phage display selection on murine C5a, with negative selection against full length human C5. The C5 antibody (BB5.1) is a mouse IgG1 antibody raised against purified mouse C581 (link). BB5.1 recognizes both the disulphide linked alpha-chain (111 kDa) and beta-chain (75 kDa) of mouse C5 and inhibits C5-dependent hemolysis. A mouse IgG1 antibody (135.8) raised against human complement component C8 was used as an isotype control82 (link). All three antibodies were each stably expressed in Chinese hamster ovary (CHO) cells and purified using single step affinity chromatography with mabselect Xtra (GE) protein A resin. Antibodies were free of endotoxin and determined to be greater than 95% pure using capillary electrophoresis.
Mabselect xtra
Manufactured by GE Healthcare
Sourced in United States
MabSelect Xtra is a protein A affinity chromatography medium designed for the purification of monoclonal antibodies. It features a highly cross-linked agarose base matrix and recombinant protein A ligand, providing high dynamic binding capacity and operational stability.
Automatically generated - may contain errors
5 protocols using mabselect xtra
1
Characterization of C5-targeting Antibodies
2
Purification of Engineered dAb Molecule
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Example 6
DOM0100, a 25 kDa (Vk−VH albudAb+TNFR1dAb) dAb molecule expressed in E. coli was purified using protein A, MabSelect Xtra from GE Healthcare packed in a 0.5×20 cm column. The flow rate was 300 cm/hr for all the steps. After equilibration with 55 mM tris-base, 45 mM acetic acid, pH7.5, cell culture filtrate was loaded on the column at 13.5 mg/mL of resin. The load titer was 1.88 mg/mL. The column was then washed using 5 column volumes of 55 mM tris-base, 45 mM acetic acid, 300 mM sodium acetate, 100 mM sodium caprylate, pH7.5. The protein was then eluted and thereafter the column was cleaned, sanitized and stored. The analysis of the elution peak gave 1,440 ppm HCPs (host cell proteins) by ELISA for a 74.9% yield. The same experiment repeated twice under the same conditions but with a high salt wash instead of the caprylate wash gave 2,398 ppm and 2,456 ppm HCPs by ELISA for a yield of 77.2% and 76.0% respectively. The effect of the chromatographic sequence evaluated in a 0.5 cm×10 cm column matching the residence time had no effect on the dynamic binding capacity up to 150 cycles. Resin selectivity was equally investigated for MabSelect and base stable MabSelect SuRe using the same chromatographic sequence and gave comparable HCP product quality.
3
Protein A Affinity Purification of IgG
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Purification of IgG using protein A based affinity chromatography was performed by applying 12 mL of 2-fold diluted HHP per run on MabSelect Xtra columns (V = 2 × 1 mL, GE Healthcare, USA) with 20 mM Tris/HCl running buffer, pH 7.4, at a flow rate of 2 mL min-1. The bound antibodies were eluted with 20 mM citric acid, pH 2.3, diafiltrated into PBS, pH 7.0, and formulated with 0.3 M glycine. A highly purified IgG sample (eIgG) was used as standard in ELISA assay and as model substrate for preliminary optimisation of pepsin digestion.
4
SARS-CoV-2 Spike Protein Expression and Purification
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Genes encoding the ecto-domain (residues 13–1,202 aa) and the RBD (residues 319–541 aa) of the SARS-CoV-2 spike glycoprotein were synthesized and cloned into the pCIW mammalian expression vector with a C-terminal 6x histidine tag. Genes encoding RBD mono-Fc and human ACE2 mono-Fc [obtained from Prof. Jason S. McLellan, University of Texas (30 (link))] were cloned into the pCIW vector. The pCIW mammalian vectors were transfected into Expi293F™ cells (Cat. no. A1435101). All the steps for expression analysis were performed according to the manufacturer’s instructions. After 5–6 days of cell cultivation, the cells were harvested by centrifugation, and the supernatant was passed through a 0.22 μm filter to remove cell debris. For His-tag purification of recombinant proteins, the Ni-NTA resin was added to the supernatant. MabSelect Xtra (Cat. no. 17-5269-02, GE Healthcare) was used as a resin for protein-A purification of Fc-fused proteins. All purification procedures were performed according to the manufacturer’s instructions. The buffer of eluted proteins was changed with phosphate-buffered saline (PBS) using Zeba Spin Desalting Columns (Cat. no. 0089892). Protein concentration was quantified using a Nanodrop 2000C spectrophotometer (Thermo Scientific) and using SDS-PAGE under non-reducing and reducing conditions.
5
Monoclonal Antibody Production Protocol
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Top-scoring heavy and light chain sequences from the corresponding NGS database searches were subcloned into the AbVec-hIgG1 and AbVec-hIgKappa expression vectors, respectively (40 (link), 41 (link)). Sanger sequencing was applied to verify the final DNA vectors. Heavy chain (500 μg) and 250 μg of the light chain DNA expression vectors were applied for cotransfection of 1 liter of human embryonic kidney 293F cells to produce Rh.33104 mAb.1 and Rh.33172 mAb.1 (as full IgG and Fab fragments). Polyethylenimine (PEI MAX, Polysciences Inc.) was used as a transfection reagent at threefold mass excess with respect to the total DNA amount. Antibodies were purified from cell supernatants using the MAbSelect Xtra (GE Healthcare Life Sciences) and CaptureSelect IgG-CH1 (Thermo Fisher Scientific) columns for IgG and Fab purification, respectively. Antibody samples were concentrated, buffer exchanged to TBS buffer (Alfa Aesar), and then subjected to SEC purification (HiLoad 16/600 Superdex S200 pg column; GE Healthcare Life Sciences).
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