Production of monospecific antibodies to a low-abundance hepatic membrane protein using nitrocellulose immobilized protein as antigen期刊界 All Journals 搜尽天下杂志传播学术成果专业期刊搜索期刊信息化学术搜索

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Production of monospecific antibodies to a low-abundance hepatic membrane protein using nitrocellulose immobilized protein as antigen

Authors:	T C Chiles T W O'Brien M S Kilberg

Institution:	1. Department of Pharmacology, The University of Tennessee Health Sciences Center, 71 S. Manassas, Memphis, TN 38103, USA;2. Department of Physiology, The University of Tennessee Health Sciences Center, 71 S. Manassas, Memphis, TN 38103, USA;3. Department of Biochemistry, Faculty of Pharmacy Cairo University, Kasr El-Aini St., Cairo 11562, Egypt;1. Laboratory of Transmissible Diseases and Biological Active Substances, Faculty of Pharmacy, University of Monastir, Avenue Avicenne, 5000 Monastir, Tunisia;2. Higher Institute of Biotechnology of Monastir, Avenue Taher Hadded, BP 74, 5000 Monastir, Tunisia;3. Research Laboratory in Biochemistry, Faculty of Medicine, University of Monastir, Avenue Avicenne, 5019, Tunisia;4. Laboratory of Bioactive Substances, Biotechnology Center in the Technopole of Borj-Cédria (CBBS), 2050 Hammam-lif, Tunisia;5. Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity, Team: Medicinal Chemistry and Natural Products, Faculty of Sciences of Monastir, University of Monastir, Avenue de l’ Environnement, 5019 Monastir, Tunisia;1. Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan;2. DHC Corporation, 2-8-21 Minami-azabu, Minato-ku, Tokyo 106-0047, Japan;3. School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakuramachi, Hachioji, Tokyo 192-0982, Japan

Abstract:	Membrane proteins from primary cultures of rat hepatocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose paper which was then dissolved in dimethyl sulfoxide and this mixture was used as a primary immunogen in rabbits. Subsequent immunizations were performed using nonsolubilized protein immobilized on nitrocellulose paper. A monospecific polyclonal antibody was generated against a specific mitochondrial membrane protein (MP-73) for which de novo synthesis appeared to be induced by amino acid starvation of the hepatocytes. A minimum of 15-20 micrograms of protein antigen was required to elicit significant antibody production. Serum antibody titer was sufficient to allow detection of MP-73 at a serum dilution of 1:2000.

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