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Selection of antibody probes to correlate protein sequence domains with their structural distribution
Author(s) -
Valle Mikel,
Muñoz Manuel,
Kremer Leonor,
Valpuesta Jose M.,
MartínezA Carlos,
Carrascosa Jose L.,
Albar Juan P.
Publication year - 1999
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.8.4.883
Subject(s) - polyclonal antibodies , antigenicity , antiserum , epitope , computational biology , biology , antibody , epitope mapping , microbiology and biotechnology , genetics
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 ø29 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.