Antibodies in the cell culture harvest were purified by capturing on a HiTrap protein G (Cytiva), followed by washing with PBS and elution with 0.1 M Glycine pH 2.7. After dialysis against 20 mM Tris pH 7.5, the sample was passed through a Q-sepharose column (Cytiva) and equilibrated with the same buffer. The flow-through was concentrated to < 5 mL and purified on a HiLoad® 16/600 Superdex® (Cytiva) using PBS as eluent. Fractions were analyzed by SDS-PAGE, SEC, and LC-MS. Selection for pooling was made to minimize aggregates, incorrectly paired molecules, and free LC. After purification, a certain amount of Hole dimer (double Hole HC with typical Fc length) was still present in the used Adu H310A -8D3 and B12 H310A -8D3 batches (Figure S3 and S4; t R = 15.2/16.3; ~ 20%).
Hitrap protein g
Manufactured by Cytiva
Sourced in United Kingdom
HiTrap protein G is a prepacked chromatography column used for the purification of antibodies and antibody fragments from various sample sources. It utilizes Protein G, a bacterial cell wall protein with a high affinity for the Fc region of immunoglobulins, to selectively bind and capture target antibodies. The column can be easily connected to a liquid chromatography system for automated, high-performance purification.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (4 mentions)
Hitrap n hydroxy succinimide activated high performance affinity column, by Cytiva (2 mentions)
Q sepharose column, by Cytiva (1 mentions)
Hiload 16 600 superdex, by Cytiva (1 mentions)
7k mwco zeba desalting column, by Thermo Fisher Scientific (1 mentions)
50k mwco amicon ultra centrifugal filter units, by Merck Group (1 mentions)
Hitrap mabselect sure, by Cytiva (1 mentions)
Mabselect, by Cytiva (1 mentions)
4 protocols using hitrap protein g
1
Affinity Purification of Antibodies
2
Generating Humanized Monoclonal Antibodies
3
Simulating Bullous Pemphigoid Complement Activation
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This experiment was performed according to the method that we established in a previous study. Auto-IgG was purified from 15 ml of mixed serum from patients with bullous pemphigoid using HiTrap Protein G and a HiTrap N-hydroxy-succinimide-activated high-performance affinity column (Amersham Biosciences, Little Chalfont, UK) coated with the BP180 NC16A domain. HaCaT cells seeded on coverslips in 6-well plates were then incubated overnight with 1 μg/ml purified pathogenic IgG at 37°C, before being incubated for 2 h with 10 μg/ml recombinant CD55 protein and 1 ml of fresh serum from healthy controls containing complement components to initiate complement activation as a simulation of the bullous pemphigoid phenotype. C3b deposition at the DEJ and cell membrane was used as a measure of the degree of complement activation.
4
Simulation of Bullous Pemphigoid Immune Response
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Auto immunoglobulin IgG was purified from 15 ml of mixed serum from BP patients using HiTrap Protein G and a HiTrap N-hydroxy-succinimide-activated high-performance affinity column (Amersham Biosciences, Little Chalfont, UK) coated with the BP180 non-collagenous 16A domain (NC16A). HaCaT cells seeded on coverslips in 6-well plates were incubated overnight with 1 μg/ml purified pathogenic IgG from BP patients, followed by addition of 10 μg/ml recombinant CD46 protein and 1 ml of fresh serum and incubation for 2 h to initiate complement activation to simulate the BP phenotype. C3 deposition at the DEJ and cell membrane was used as a measure of the degree of complement activation.
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