Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila
Figure 2
BN-PAGE of solubilized T. thermophila mitochondrial membranes.
(A) BN gel (3%–10%) run with T. thermophila mitochondria (1 mg) solubilized with digitonin (5 µg of detergent/µg of protein), and stained with colloidal Coomassie blue. (B) In-gel ATPase staining of a BN gel strip of digitonin-solubilized T. thermophila mitochondria. The gel was incubated overnight (8–12 h) and was briefly (2 min) washed with 10% acetic acid to remove excess lead precipitate on the surface. (C) 2-D BN/BN-PAGE. The first dimension was completed as in (A) and a strip was excised and briefly soaked in cathode buffer containing 0.03% docecyl maltoside (the strip shown here above the 2-D gel is a second strip cut from the same 1-D BN-PAGE that was stained with Coomassie blue; the image of the strip was cropped below the position of band 3). The second dimension was a 4%–12% gradient BN-PAGE run with 0.03% dodecyl maltoside in the cathode buffer (see Materials and Methods). The band 1 (V2, I+III2, II2) separated into two spots designated as spot 1 and 2. The band 2 (V2, I+III2) separated into two spots designated as spot 3 and 4, while band 3 (III2) ran as a single spot, labeled as spot 5. The image of the 2-D gel was cropped on the right side so that most of the material running below band 3/spot 5 is not shown.