Determination of Protein Expression in Transgenic Mice

Actin contributes to the movement and shape of eukaryotic cells and maintains organized structure necessary for the function of striated muscle. Actin is regulated by a variety of accessory proteins including actin capping proteins (CP). CP is composed of an alpha (α) and beta (β) subunit. In eukaryotes, there are three alpha subunits, αl, α2, α3, and three beta subunits, βi, β2, and β3. To elucidate the function of the beta subunits, transgenic mice were generated that lead to a decrease in expression of the pi subunit. In preliminary studies, the level of CPβl in three month old transgenic mice hearts was reduced approximately two fold relative to wildtype hearts. To confirm and extend these studies, we determined the level of expression of CPβl, CPβ2, actin and CPα in transgenichearts. Hearts from transgenic and wildtype mice at 3, 6, 9, and 12 months post gestation were pulverized in liquid nitrogen, solubilized in sodium dodecyl sulfate (SDS) sample buffer, and the proteins separated via SDS polyacrylamide gel electrophoresis. The separated proteins were quantitated via densitometry to ensure a constant load of protein. The proteins were transferred to nitrocellulose and probed with antibodies including anti-actin, anti-pan α, anti-βl, and anti-β2. The antibody:protein complex was visualized through the use of a secondary antibody labeled with alkaline phosphatase which allowed for a visible precipitate upon reaction with its substrate. The immune complexes were quantitated via densitometry. This information will be useful to correlate the level of expression with the phenotypes of the transgenic mice