A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue
A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue
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DOI：
10.1002/pmic.200400892
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发表时间：
2005-01-01
期刊：
影响因子：
3.4
通讯作者：
Nothwang, HG
中科院分区：
文献类型：
--
作者：
Guillemin, I;Becker, M;Nothwang, HG
亚细胞定位是蛋白质组分析的一个重要方面，尽管经常被忽视。通常，蛋白质的亚细胞位置决定细胞和组织的功能。在这里，我们提出了一个强大的和通用的预分离哺乳动物细胞和组织，这是适当的，微量样品量的协议。该协议产生三个部分：细胞核，细胞质，和组合的膜和细胞器部分。通过对5种标记蛋白的免疫印迹分析以及通过二维凝胶电泳和质谱对43种蛋白的分析，证明了亚细胞特异性和组分的组成。为了覆盖所有蛋白质种类，进行常规二维和苄基二甲基-正十六烷基氯化铵-十二烷基硫酸钠（16-BAC-SDS）凝胶电泳。完整的膜蛋白和强碱性核组蛋白只在16-BAC-SDS凝胶电泳系统中检测到，证实了其用于蛋白质组分析的有用性。除一种蛋白质外，所有蛋白质均符合所分析组分的相应亚细胞组成。两者合计，这些数据使我们的亚细胞prefractionation协议的一个有吸引力的替代其他prefractionation方法，这是基于较少的生理蛋白质的特性。
Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate,for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.