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Cell fractionation can be used to determine the localization and trafficking of proteins between cellular compartments such as the cytosol, mitochondria, and nuclei. Subcellular fractionation is usually performed immediately after tissue dissection because freezing may fragment cell membranes and induce organellar cross-contamination. Mitochondria are especially sensitive to freezing/thawing and mechanical homogenization. We proposed a protocol to improve the retention of soluble proteins in the mitochondrial fraction obtained from small amounts of frozen skeletal muscle. Fifty milligrams of the red portion of gastrocnemius muscle from Wistar rats were immediately processed or frozen in liquid nitrogen and stored at -80 °C for further processing. We compared the enrichment of subcellular fractions from frozen/fresh samples obtained with the modified protocol with those obtained by standard fractionation. Western blot analyses of marker proteins for cytosolic (alpha-tubulin), mitochondrial (VDAC1), and nuclear (histone-H3) fractions indicated that all of the procedures resulted in enriched subcellular fractions with minimal organellar cross-contamination. Notably, the activity of the soluble protein citrate synthase was higher in the mitochondrial fractions obtained with the modified protocol from frozen/fresh samples compared with the standard protocol. Therefore, our protocol improved the retention of soluble proteins in the mitochondrial fraction and may be suitable for subcellular fractionation of small amounts of frozen skeletal muscle samples.
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