Fig. 1

Optimization of the digitonin concentration for mitochondrial polysome isolation. To improve the extraction efficiency of the membrane-bound mitochondrial ribosomes, the detergent digitonin was added to the extraction buffer. The extraction efficiency of mitochondrial ribosomes was checked by hybridization to a specific probe for the mitochondrial cox1 gene (a). As the original protocol had been optimized for the isolation of plastidial polysomes, the influence of increased digitonin concentrations on plastidial ribosomes was determined by using a psbA gene specific probe as a control (b). Blots under the heading “mitochondrial probe cox1” (a) and “plastid probe psbA” (b) indicate two sets of parallel experiments for the optimization of digitonin concentrations. Blots under the  label “minus puromycin” indicate the polysome purification experiment and those under the label “plus puromycin” are controls to identify light and heavy polysomal fractions. In both a and b set of blots (i) represents polysome isolation procedure using a protocol suitable for plastidial polysomes where lanes 1–10 represent the different polysomal fractions. (ii) Polysome isolation by supplementing the extraction buffer with 50 mg/ml digitonin. (iii) Polysome isolation by the addition of 60 mg/ml digitonin to the extraction buffer. (iv) Polysome isolation using 75 mg/ml digitonin in the extraction buffer