Figure 4.

Western Blot of plasma membrane H⁺-ATPase from K. lactis. The plasma membranes (PM) were isolated from the yeast K. lactis by differential centrifugation, and the H⁺-ATPase was isolated (Figure 2) by ultracentrifugation on a trehalose gradient [16]. After that, size exclusion chromatography (Superose^® 6 column) was performed and two high absorbing (λ= 280 nm) peaks (Peak 1 and Peak 2) containing the H⁺-ATPase hexamer were eluted (Figure 2 and Figure 3) when loading 3 mg protein. H⁺-ATPase samples (PM, Peak 1, and Peak 2) were subjected to SDS-PAGE; a sample of the supernatant (S) from PM purification was included as a negative control. The Western Blot (WB) assay was performed after protein electrophoretic transfer from the denaturing gel to a PVDF membrane and immunoblotted using polyclonal antibodies generated against the recombinant C-terminal domain of H⁺-ATPase from Saccharomyces cerevisiae. The immunoreactivity was evidenced using the Immuno Blot^® assay kit. The lane of molecular weight standards (MW std) is included and is stained with Coomassie blue.