Human FcγRI, FcγRIIa_R131 and FcγRIIIa_V158 receptors used in this work were expressed in-house in HEK293F for FcγRI, FcγRIIa_R131 (transient expression) and in CHO cells for FcγRIIIa_V158, respectively. The CHO DG44 cell line was stably transfected [36 (link)]. Purification of the receptor was achieved by affinity chromatography using Ni Sepharose High Performance material (GE Healthcare, Munich, Germany) followed by elution with 300 mM imidazole (Sigma, Munich, Germany) an case of FcγRI, FcγRIIa_R131 and 100 mM imidazole in case of FcγRIIIa_V158, and a size exclusion chromatography on Superdex 200 26/60 column (GE Healthcare, Munich, Germany) with PBS pH7.4 (FcγRI, FcγRIIa_R131) or 2 mM MOPS, 150 mM NaCl, 0.02% Tween 20 pH 7.0 buffer (FcγRIIIa_V158). Fcγ receptors were biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions (Bulk BIRA, Avidity LLC, Denver, CO, USA) and dialyzed at 4°C over night to remove excess of biotin. The product quality was characterized by standard methods. The glycosylation pattern of FcγRI and FcγRIIa is not relevant for the antibody interaction, and was not addressed in detail. Analysis of the glycosylation pattern of FcγRIIIa was described previously [36 (link)].
Bulk bira
Manufactured by Avidity
Sourced in United States
Bulk BIRA is a laboratory equipment designed for the preparation and processing of biological samples. It serves as a versatile tool for researchers and scientists working in the field of biotechnology, molecular biology, and related disciplines.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using bulk bira
1
Recombinant FcγR Expression and Purification
2
Preparation of C1q Affinity Column
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Example 2
Preparation of C1q Affinity Column
Single chain C1q fusion polypeptide with an Avi Tag in 2 mM MOPS buffer comprising 125 mM NaCl and 0.02% Tween, adjusted to pH 7.2, and supplemented with 1 tablet Complete protease inhibitor (cOmplete ULTRA Tablets, Roche Diagnostics GmbH, Mannheim, Germany) in 3 ml PBS was biotinylated using the biotinylation kit from Avidity according to the manufacturer instructions (Bulk BIRA, Avidity LLC, Denver, Colo., USA). Biotinylation reaction was done at room temperature overnight. To separate the ligase Ni-Sepharose chromatography (see above) was repeated. The modified polypeptide was dialyzed against 50 mM sodium phosphate buffer comprising 500 mM NaCl, pH 7.2 at 4° C. overnight to remove imidazole.
One gram streptavidin sepharose (GE Healthcare) was added to the biotinylated and dialyzed polypeptide (for standard analytical applications 3 mg of C1q were chosen) and incubated for two hours with shaking. The receptor derivatized sepharose was filled in a 1 ml Tricom 5/50 column (GE Healthcare).
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