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Selection of antibody probes to correlate protein sequence domains with their structural distribution

Published online by Cambridge University Press:  01 April 1999

MIKEL VALLE
Affiliation:
Department of Macromolecular Structure, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
MANUEL MUÑOZ
Affiliation:
Department of Immunology and Oncology, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
LEONOR KREMER
Affiliation:
Department of Immunology and Oncology, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
JOSE M. VALPUESTA
Affiliation:
Department of Macromolecular Structure, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
CARLOS MARTÍNEZ-A.
Affiliation:
Department of Immunology and Oncology, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
JOSE L. CARRASCOSA
Affiliation:
Department of Macromolecular Structure, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
JUAN P. ALBAR
Affiliation:
Department of Immunology and Oncology, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, 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 φ29 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.

Type
Research Article
Copyright
© 1999 The Protein Society

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