New method of plant mitochondria isolation and sub-fractionation for proteomic analyses

A new method for the isolation and sub-fractionation of mitochondria from plant tissues is described. This method was used for the analysis of proteins isolated from a pure total mitochondrial fraction and of protein complexes obtained from two mitochondrial sub-fractions, membrane-bound and matrix, from in vitro cultivated tobacco pollen tubes. The method comprised: a new plant tissue homogenization procedure; differential centrifugation of the homogenates in a Percoll gradient; mitochondria immobilization together with Percoll removal by filtration through an uncharged nylon membrane. Mitochondria trapped on the nylon membrane were used for (1) rapid protein extraction; (2) quantitative phenol–chloroform protein extraction; (3) mitochondria sub-fractionation into membrane-bound and matrix fractions. The new method was found to be more efficient than previously published protocols, requiring the use of as little as 180 mg of fresh plant material. It is very fast, reliable, and suitable for proteomic analyses of plant organs and tissue samples with low biomass. © 2004 Elsevier Ireland Ltd. All rights reserved.