Polyclonal Antibody Production and Validation for CCDC88B

Polyclonal rabbit anti-human CCDC88B antiserum was prepared as we have previously described^{5 (link),34 (link)}. A recombinant protein consisting of glutathione-S-transferase (GST) fused in-frame to a CCDC88B segment corresponding to human amino acid positions 650 to 769, was expressed in E. coli BL21, followed by affinity purification with glutathione-agarose beads (GE Healthcare). Antisera were raised in New Zealand white rabbits using purified protein (0.5 mg per rabbit per injection) emulsified in Freund’s incomplete adjuvant. Affinity purification of the anti-CCDC88B antibody^{35 (link)} was using a recombinant CCDC88B protein (positions 650 to 769) comprising a poly-histidine tail (His)6 fused in-frame at its N terminus, and purified by chromatography onto Ni-NTA agarose (Qiagen). The immobilized protein was used to capture the anti-CCDC88B fraction of the hyperimmune serum, which was then released by washing with imidazole-containing buffer^{34 (link)}. The specificity of the anti-CCDC88B antibody was tested by western blotting total cell extracts from HEK293T control cells and HEK293T cells (ATCC, CRL-11268) stably expressing a full-length human CCDC88B.

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Fodil N., Moradin N., Leung V., Olivier J.F., Radovanovic I., Jeyakumar T., Flores Molina M., McFarquhar A., Cayrol R., Bozec D., Shoukry N.H., Kubo M., Dimitrieva J., Louis E., Theatre E., Dahan S., Momozawa Y., Georges M., Yeretssian G, & Gros P. (2017). CCDC88B is required for pathogenesis of inflammatory bowel disease. Nature Communications, 8, 932.

Publication 2017

glutathione-agarose beads Ni-NTA agarose HEK293T cells

Affinity purification Agarose Amino acid Antibody specificity Antisera Cells Cells extracts Chromatography E coli Frame Freund adjuvant Glutathione Glutathione s transferase Human Imidazole New zealand white rabbits Poly histidine Protein Rabbit Recombinant protein Serum Tail

Corresponding Organization : McGill University

Other organizations : Centre Hospitalier de l’Université de Montréal, Université de Montréal, Icahn School of Medicine at Mount Sinai, RIKEN Center for Integrative Medical Sciences, University of Liège, Leona M. and Harry B. Helmsley Charitable Trust

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Variable analysis

independent variables

Purified recombinant protein consisting of GST fused to a CCDC88B segment (amino acid positions 650 to 769)

dependent variables

Antibody specificity, as tested by western blotting of total cell extracts from HEK293T control cells and HEK293T cells stably expressing full-length human CCDC88B

control variables

HEK293T control cells (without CCDC88B expression)

controls

Positive control: HEK293T cells stably expressing full-length human CCDC88B
Negative control: HEK293T control cells (without CCDC88B expression)

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