Selection of antibody probes to correlate protein sequence domains with their structural distribution |
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| Authors: | Valle M Muñoz M Kremer L Valpuesta J M Martínez-A C Carrascosa J L Albar J P |
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| Affiliation: | Department of Macromolecular Structure, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, Spain. |
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| Abstract: | We propose a new approach that permits correlation of specific domains defined by their primary sequence with their location in the structure of complex macromolecular aggregates. It is based on the combination of well-established structural analysis methods that incorporate the use of overlapping peptides on cellulose membranes for the isolation and purification of specific antibodies from a polyclonal antiserum. Monospecific antibodies to the connector protein of bacteriophage phi29 were isolated from polyclonal antisera using a new development of the spotscan method. These antibodies can be purified in quantities that allow antigenicity testing in enzyme-linked immunosorbent assays, Western blotting and immunoprecipitations, demonstrating the specificity of this isolation procedure. This approach has allowed us to generate direct antibody probes for immunoelectron microscopy mapping of different connector protein domains in a low resolution three-dimensional epitope map. |
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| Keywords: | antibody purification bacteriophage ø29 connector protein cellulose membrane-bound peptides epitope mapping spot synthesis |
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