Developing Antibodies to Synthetic Peptides Based on Comparative DNA Sequencing of Multigene Families
By ROGER H. SAWYER. TRAVIS C. GLENN, JEFFREY O. FRENCH, and LOREN
W. KNAPP
Abstract
Using antisera to analyze the expression of specific gene products is
a common procedure. However, in multigene families, such as the ß-keratins
of the avian integument where strong homology exists among the scale (Scßk),
claw (C1ßK), feather (FßK), and feather-like (F1ßK)
subfamilies, determining the cellular and tissue expression patterns of
the subfamilies is difficult because polyclonal antisera produced from
anyone protein recognize all family members. Traditionally, researchers
produced and screened multiple monoclonal antisera produced from the proteins
of interest until an antiserum with sufficient specificity could be obtained.
Unfortunately, this approach requires a lot of effort, and once obtained,
such antisera may have limited applications. Here, we present procedures
by which comparative DNA sequences of members from the ß-keratin
multigene family were translated
and aligned to identify amino acid domains that were conserved within
the FßK subfamily, but which were divergent from the other subfamilies.
A synthetic 23-mer peptide with the conserved amino acid sequence was
generated and used to produce a polyclonal antiserum that recognizes only
the FßK subfamily of proteins. Western blot analysis and confocal
microscopy with this antiserum are now providing valuable new insights
concerning the developmental and evolutionary relationships between the
scale, claw, and feather proteins found in birds. This represents a powerful
new approach combining techniques from molecular evolution and developmental
biology to study the expression and evolution of specific members of multigene
families.
SREL Reprint #2869
Sawyer, R. H., T. C. Glenn, J. O. French and L. W. Knapp. 2005. Developing antibodies to synthetic peptides based on comparative DNA sequencing of multigene families. Methods in Enzymology 395:636-652.
