Western Blot Analysis for Detection of the A2 Subunit of the (Na+, K+)-Atpase in Mouse Brain

Onofry, Kevin, Michael Janice, and Mary Caspers

The (Na+, K+)-ATPase catalyzes the active transport of Na+ and K+ across cell membranes and helps to reestablish ion balance after a neuron has fired. It is composed of α and β subunits; three isoforms of the α subunits exist in brain. Previous work in this laboratory has detected the presence of the α2 and α3 subunits of the (Na+, K+)-ATPase in mouse brain sections (24 micron) using [3H]ouabain binding to the subunits. In order to specifically identify the location of the α2 subunits, work began to optimize an indirect immunohistochemical (IHC) technique using goat polyclonal IgG antibody directed against the α2 subunit of the (Na+, K+)-ATPase, and rabbit anti-Goat IgG conjugated to horseradish peroxidase (HRP) directed against the primary antibody. Visualization was accomplished using a luminal substrate and CL-XPosure X-ray film. The inability to obtain reproducible results using IHC led to the use of western blots (10% gel) using the same primary and secondary antibodies. The results are much more promising showing staining of the α2 subunit (~113 kDa); however, there is a significant amount of background staining. We will continue to test different concentrations of primary and secondary antibodies along with different blocking and washing techniques in order to reduce the amount of non-specific staining. When optimal conditions have been ascertained, we will apply this procedure to brain tissue from wild type and heterozygous α2 knockout mice. We gratefully acknowledge the Department of Chemistry and Biochemistry, University of Detroit Mercy, for supporting this project.