PDF(Pubmed)
Abstract：
REAP+ is an enhanced version of the rapid, efficient, and practical (REAP) method designed for the isolation of nuclear fractions. This improved version, REAP+, enables fast and effective extraction of mitochondria, cytoplasm, and nuclei. The mechanical cell disruption process has been optimized to cerebral tissues, snap-frozen liver, and HT22 cells with remarkable fraction enrichment. REAP+ is well-suited for samples containing minimal protein quantities, such as mouse hippocampal slices. The method was validated by Western blot and marker enzyme activities, such as LDH and G6PDH for the cytoplasmic fraction and succinate dehydrogenase and cytochrome c oxidase for the mitochondrial fraction. One of the outstanding features of this method is its rapid execution, yielding fractions within 15 min, allowing for simultaneous preparation of multiple samples. In essence, REAP+ emerges as a swift, efficient, and practical technique for the concurrent isolation of nuclei, cytoplasm, and mitochondria from various cell types and tissues. The method would be suitable to study the multicompartment translocation of proteins, such as metabolic enzymes and transcription factors migrating from cytosol to the mitochondria and nuclei. Moreover, its compatibility with small samples, such as hippocampal slices, and its potential applicability to human biopsies, highlights the potential application in medical research.
摘要：
REAP+是快速的增强版本，高效,和实用（REAP）方法设计用于分离核部分。这个改进的版本，REAP+,能够快速有效地提取线粒体，细胞质，和原子核。机械细胞破碎过程已针对脑组织进行了优化，速冻肝脏,和HT22细胞显著富集。REAP+非常适合含有最少蛋白质量的样品，如小鼠海马片。方法经Westernblot和标记酶活性验证,例如用于细胞质部分的LDH和G6PDH以及用于线粒体部分的琥珀酸脱氢酶和细胞色素C氧化酶。该方法的突出特点之一是其快速执行，在15分钟内产生馏分，允许同时制备多个样品。实质上,REAP+出现迅速，高效,以及同时分离原子核的实用技术，细胞质，和线粒体来自各种细胞类型和组织。该方法适用于研究蛋白质的多室易位，如代谢酶和转录因子从细胞质迁移到线粒体和细胞核。此外，它与小样品的兼容性，比如海马切片，以及它对人体活检的潜在适用性，突出了在医学研究中的潜在应用。
