SREL Reprint #2869

 

 

 

Developing Antibodies to Synthetic Peptides Based on Comparative DNA Sequencing of Multigene Families


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.

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