差速离心
蛋白质组
分馏
细胞分离
蛋白质组学
高尔基体
样品制备
内质网
离心
化学
质谱法
蛋白质亚细胞定位预测
萃取(化学)
蛋白质纯化
色谱法
串联质谱法
生物化学
膜
基因
作者
Brian Cox,Andrew Emili
出处
期刊:Nature Protocols
[Springer Nature]
日期:2006-11-01
卷期号:1 (4): 1872-1878
被引量:297
标识
DOI:10.1038/nprot.2006.273
摘要
We have shown that sample fractionation is an effective method for increasing the detection coverage of the proteome of complex samples, such as organs, by mass-spectrometric techniques. Further fractionating a sample based on subcellular compartments can generate molecular information on the state of a tissue and the distribution of its protein components. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs or tissues for subsequent analysis by mass spectrometry. It uses differential centrifugation in density gradients to isolate nuclear, cytosolic, mitochondrial and mixed microsomal (Golgi, endoplasmic reticulum, other vesicles and plasma membrane) fractions. Once the fractions are isolated, they are extracted for protein and the samples can then be frozen for processing and analysis at a later date. The procedure can typically be completed in 5 h.
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