Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channel subunits

• Published in: Brain research. Brain research protocols Vol. 1; no. 3; pp. 307 - 319
• Main Authors: Beattie, Ruth E, Volsen, Stephen G, Smith, Dawn, McCormack, Alison L, Gillard, Samantha E, Burnett, J.Paul, Ellis, Steven B, Gillespie, Alison, Harpold, Michael M, Smith, William
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.1997
• Subjects: Animals; Antibodies - immunology; Antibodies - isolation & purification; Antibody Specificity; Antibody to fusion protein; Antigens - immunology; Blotting, Western; Calcium channel; Calcium Channels - immunology; Calcium Channels - physiology; Cell Line; Characterisation of specific antibody; Electrophysiology; Enzyme-Linked Immunosorbent Assay; Escherichia coli - metabolism; Fluorescent Antibody Technique; Fusion protein; Humans; Immunization; Neurons - metabolism; Purification of specific antibody; Rabbits; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - metabolism; Purification of specific antibody; Fusion protein; Characterisation of specific antibody; Calcium channel; Antibody to fusion protein
• ISSN: 1385-299X
• DOI: 10.1016/S1385-299X(97)00004-4

Neuronal voltage-dependent calcium channels (VDCCs) each comprising of α 1, α 2 δ, and β subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation [3, 11]. Studies have shown the expression of several α 1 and β subtypes within neuronal tissue [8]. The comparative distribution of these in normal human brain is largely unknown. The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain. Previous studies have defined DNA sequences specific for each subunit [15]. Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E. coli. Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns. The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding α 1, α 2 δ and β subunits.