Zebrafish Protein & Antibody Core

PI. Brian K. Kay, Ph.D.
Professor & Head, Department of Biological Sciences
University of Illinois, Chicago
Frank Collart, Co-Investigator
Argonne National Lab

BACKGROUND
More and more researchers have turned to the zebrafish as their favorite vertebrate model system because of its versatility. Owing to its popularity, the zebrafish is one of the vertebrates whose genome is currently being sequenced. Despite the wealth of information gathered on the zebrafish since its contributions to science beginning in the early 1980s, the availability of zebrafish proteins and antibodies has been lacking. The Zebrafish Protein & Antibody Core will take the first formal step in creating a national resource for the production of zebrafish proteins and their respective antibodies for use by the zebrafish research community.   The Zebrafish Protein and Antibody Core focuses on the production of zebrafish proteins and protein fragments, the generation of antibodies to those proteins, and the fabrication of arrayed proteins and affinity reagents

Antibody Production.
Antibodies are useful for detecting and quantitating endogenous proteins in a cell or organism. A phage library, consisting of the variable domain of the IgG light chain (VL) linked to the variable domain of the IgG heavy chain (VH) in a single polypeptide and displaying 6 billion different human scFvs, is used for affinity selections with proteins provided by the ANL Group. Two to three rounds of affinity selection are sufficient to isolate binding phage from this library.  After the antibody is confirmed for affinity and specificity, the scFv coding region is transferred into an expression vector and can be directly used in laboratories of the zebrafish research community

Protein Production.
Proteins are the workhorse molecules of all biological systems and comprehensive knowledge of their biochemical properties and cellular function are essential to an understanding of cellular and physiological processes. The goal of the Protein Production Group at Argonne National Laboratory is the development of effective strategies for the production of zebrafish proteins. A majority of the expression and solubility trials for the target proteins used an E. coli expression system, however, targets that fail are prioritized and a subset cloned and expressed using a eukaryotic expression system.