Preparation of Module-Specific Antibodies Against CCN Family Members

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1489; p. 115
• Main Authors: Kubota, Satoshi, Takigawa, Masaharu
• Format: Journal Article
• Language: English
• Published: United States 01.01.2017
• Subjects: Animals; Antibodies - immunology; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - immunology; Antibody Formation; CCN Intercellular Signaling Proteins - chemistry; CCN Intercellular Signaling Proteins - genetics; CCN Intercellular Signaling Proteins - immunology; Cell Line; Epitopes - immunology; Humans; Hybridomas; Mice; Peptide Fragments - immunology; Plasmids - genetics; Plasmids - immunology; Rabbits; Recombinant Proteins - immunology; Monoclonal antibodies; CCN family; DNA immunization; Modules; CTGF
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-6430-7_12

Specific antibodies against biomolecules are conventional, but robust tools for the structural and functional analysis of target molecules. Since CCN family proteins are composed of four distinct modules that together determine the functionalities as full-length molecules depending upon extracellular microenvironment, specific antibody against independent modules are quite useful in CCN family research. Three distinct strategies are considerable for raising antibodies specific to four modules: IGFBP, VWC, TSP1, and CT modules. In the first strategy, full-length CCN family proteins are used to immunize mice to obtain a number of hybridoma clones producing different monoclonal antibodies, which are to be characterized to locate the epitopes in particular modules. Second methodology is a straightforward one, in which each modular protein fragment or synthetic peptide is prepared and is used for the immunization of animals independently. Finally, DNA immunization technology is recently known to be useful in developing module-specific antibodies against CCN family proteins as well. Preparation of antibodies is a quite classical and established technique, and thus nowadays is managed mostly by professional and commercial facilities. Therefore in this chapter, essentials of each strategy are introduced, rather than experimental details in each process.