Selection of antibody probes to correlate protein sequence domains with their structural distribution.
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.
Valle
M
M
Department of Macromolecular Structure, Centro Nacional de Biotecnología, CSIC, Campus de la Universidad Autónoma de Madrid, Cantoblanco, Spain.
Muñoz
M
M
Kremer
L
L
Valpuesta
J M
JM
Martínez-A
C
C
Carrascosa
J L
JL
Albar
J P
JP
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry* / Q000302:isolation & purification; D017103:Bacillus Phages / Q000737:chemistry; D001412:Bacillus subtilis / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D005456:Fluorescent Dyes; D015151:Immunoblotting; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary
D000906:Antibodies; D000939:Epitopes; D005456:Fluorescent Dyes