A critical comparison between two classical and a kit‐based method for mitochondria isolation

Abstract

Numerous protocols for isolation of mitochondria are available. Here, three methods for the isolation of intact mitochondria from mouse liver tissues are compared with regard to yield, purity and activity. Mitochondria were isolated by sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method. Our analyses show that the sophisticated (and most expensive) free-flow electrophoresis method enables isolation of intact mitochondria with an enrichment of approximately 70%. Using the classical density centrifugation method is very laborious and time-consuming, but delivers about 57% intact mitochondria. Using standard laboratory equipment in a quick and simple procedure, the kit provides approximately 50% intact mitochondria, suitable for most standard investigations.

Succinate Dehydrogenase of Saccharomyces cerevisiae – The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives

“…SDH activities can be measured in vitro in cell lysates or in mitochondrial fraction as well as in situ in individual cells. Since SDH is bound to the inner membrane, it is easily isolated along with the mitochondria by different techniques: sucrose density gradient ultracentrifugation, free-flow electrophoresis or a commercially available kit-based method [20]. The mitochondrial fraction is the source of the enzyme.…”

The Current State of the Golgi Proteomes

“…Given the success achieving high purity fractions (Taylor et al, 1997a) and subcompartmental resolution of Golgi structures (Hartelschenk et al, 1991), it is surprising that a corresponding proteomic study has not been undertaken in rats. FFE was foremost amongst techniques compared for purification of mouse mitochondria (Hartwig et al, 2009) whilst impressive results were achieved after separating populations of PM vesicles (Cutillas et al, 2005), suggests that FFE still has much to contribute to both Golgi and other subcellular proteomes. In Arabidopsis, the Golgi proteome was characterized from only two to three fractions out of approximately 15 fractions over which Golgi proteins were detected.…”

Proteomic approaches to the study of renal mitochondria

“…Using free flow electrophoresis, high purity of mitochondria can be reached 37 . Low yield and the necessity for specialized equipment are major drawbacks of free flow electrophoresis 36 . Mitochondria can be isolated with enrichment and purity comparable to ultracentrifugation methods using magnetic microbeads 38 .…”

“…Further, subcellular localization of identified proteins can be checked in databases of mitochondrial proteins 34 or by systems that predict subcellular localization 35 . Commercially available kits based on differential centrifugation can simplify and speed up the separation and could be an alternative approach when limited amounts of sample are available or for processing large numbers of samples 36 . Alternatives to differential centrifugation are separation of mitochondria using magnetic microbeads of free flow electrophoresis.…”