Isolation of Plant Mitochondria Using Affinity Purification.

Mitochondria are highly dynamic organelles that are vital for eukaryotic cellular metabolism. Mitochondria account only for a small fraction of the total cell volume. As such, intact organelle isolation is a basic requirement to study mitochondrial biochemistry and physiology. However, established isolation procedures are tedious, and require a substantial amount of tissue material to start with. We addressed this matter and developed a single step purification for Arabidopsis thaliana (Arabidopsis) mitochondria. Ubiquitous expression of an 3xHA-sGFP-TOM5 fusion protein in the outer mitochondrial membrane allows affinity purification by immunocapture in a single step. We here provide a detailed step-by-step protocol for rapid immunocapture of intact and physiologically active mitochondria from Arabidopsis plants.