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.

中文翻译：

---

冷冻骨骼肌样品的亚细胞分级分离。

细胞分级分离可用于确定蛋白质在细胞区室（如细胞质，线粒体和细胞核）之间的定位和运输。亚细胞分级通常在组织解剖后立即进行，因为冷冻可能会破碎细胞膜并引起细胞器交叉污染。线粒体对冷冻/解冻和机械均质化特别敏感。我们提出了一项协议，以改善可溶性蛋白在从少量冷冻骨骼肌中获得的线粒体部分中的保留。立即对来自Wistar大鼠的50毫克腓肠肌红色部分进行处理或将其冷冻在液氮中，并储存在-80°C下进行进一步处理。我们比较了通过修改后的协议获得的冷冻/新鲜样品中亚细胞部分的富集与通过标准分离获得的亚细胞部分的富集。对胞浆（α-微管蛋白），线粒体（VDAC1）和核（组蛋白-H3）级分的标记蛋白进行蛋白质印迹分析表明，所有操作均产生了富集的亚细胞级分，且细胞器交叉污染最小。值得注意的是，与标准方案相比，用改良方案从冷冻/新鲜样品得到的线粒体级分中的可溶性柠檬酸合酶的活性更高。因此，我们的协议改善了线粒体部分中可溶性蛋白的保留，可能适用于少量冷冻骨骼肌样品的亚细胞分离。