Quantifying the Immunoreactivity of Polyclonal IGG and IGY

Actin, a filament found in the cytoplasm found in all eukaryotic cells, contributes to cell shape, cell mobility, and to the organization of certain tissues such as striated muscle. Actin is regulated by a variety of proteins, including actin capping protein (CP). CP is composed of two subunits, an alpha and a beta subunit. One goal of Dr. Hart\u27s laboratory is to determine if the alpha subunits have similar or distinct functions in cells and tissues. As a first step towards accomplishing this goal, we will determine the location of the alpha subunits in cells/tissues using antibodies specific for each alpha isoform. The goal of our research was to characterize recently generated chicken and rabbit anti-Dl and 2 IgG and IgY antibodies, quantifying their immonoreactivity. Murine hearts were removed, flash frozen, and the tissue solubilized. The proteins were separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to Nitrocellulose (NC) for subsequent Western Blot analysis. The immobilized proteins were allowed to react with dilutions of the antibodies and visualized with a secondary antibody labeled with alkaline phosphatase. The reactive titers of both the rabbit anti-D 1 IgG and the chicken anti 2 IgG and IgY antibodies were determined, providing an initial characterization of the newly generated antibodies and suggesting an approximate working dilution for subsequent studies