Rapid, quantitative isolation of mitochondria from rat liver using Ficoll gradients in vertical rotors

A method for the rapid, quantitative isolation of rat liver mitochondria has been developed. The method consists of placing portions of whole liver homogenate on linear Ficoll gradients followed by reorienting rate-zonal centrifugation in a vertical rotor. Fractions collected from the gradient were analyzed for protein, cytochrome oxidase, and a number of marker enzymes to determine mitochondrial location and the extent of their contamination with other cellular organelles. In the fraction designated as the mitochondrial peak, 85% of the total cytochrome oxidase activity was recovered. Contamination with other cellular organelles ranged from 7.07 to 14.05%. The respiratory control ratio of the isolated mitochondria was 8.10, indicating they were well coupled.