Figure 8. 2D BN/SDS-PAGE separation of native astrocytoma mitochondria protein extracts.

Two-dimensional separation was performed as described in the Materials and Methods, and the PVDF membrane was first immunoblotted for the BK_Ca channel β4 subunit (below, Coomassie staining panel). Next, the PVDF membrane was immunoblotted for the subunits of individual respiratory chain complexes (below the BK_Ca β4 panel). The BN-PAGE was calibrated based on the location of mitochondrial respiratory chain complexes that were isolated from rat heart mitochondria (above the panel for the blue native PAGE of mitochondria from astrocytoma cells). In the native astrocytoma lysate, mitochondria BK_Ca β4 co-localized with subunit I of cytochrome c oxidase. M, the monomeric form of cytochrome c oxidase; D, the dimeric form of cytochrome c oxidase; Sc₁ and Sc₂, complexes with higher molecular weights containing cytochrome c oxidase. A typical immunoblot from three separate experiments is shown.