| Cell Fractionation of Pseudomonas aeruginosa
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Author:
Date:
2013-10-05
[Abstract] Pseudomonas aeruginosa is a Gram negative bacterium. Separating the cell envelope compartments enables proteins to be localized to confirm where in the cell they function. Cell fractionation can also provide a first step in a protein purification strategy (Williams et al., 1998). This protocol has been designed to obtain the different fractions of P. aeruginosa, namely the inner membrane, outer membrane, cytoplasmic and periplasmic compartments. Specific detection of the arginine specific autotransporter (AaaA) (Luckett et al., 2012) in the outer membrane of P. aeruginosa has been performed using this protocol.
[摘要] 铜绿假单胞菌是革兰氏阴性细菌。 分离细胞包膜区室使得蛋白质能够被定位以确认它们在细胞中的何处起作用。 细胞分级还可以提供蛋白质纯化策略的第一步（Williams等人，1998）。 该方案已经设计成获得不同比例的P。 铜绿假单胞菌，即内膜，外膜，细胞质和周质区室。 在P的外膜中特异性检测精氨酸特异性自转运体（AaaA）（Luckett等人，2012）。 已经使用该协议进行了铜绿假单胞菌。
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| Isolation of Inner Membrane Vesicles from Escherichia coli by Using an Affinity Tag
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Author:
Date:
2012-10-20
[Abstract] This protocol was developed in a project aimed to identify the inner membrane proteins localizing to cell poles in Escherichia coli (E. coli). By using a known polar protein Tar as a tag, we isolated pole-derived inner membrane vesicles by affinity capture. The specificity of the polar vesicle isolation was confirmed by mass spectrometry that identified more than one hundred proteins, most of which are known inner membrane proteins, including other known polar proteins. This protocol, or if adapted properly by choosing other affinity targets, is well suited to isolate other membrane domains of interest for identification of proteins or lipid composition.
[摘要] 该方案是在旨在鉴定定位于大肠杆菌（E.coli）中的细胞极的内膜蛋白的项目中开发的。 通过使用已知的极性蛋白质Tar作为标签，我们通过亲和捕获分离杆来源的内膜囊泡。 极性囊泡分离的特异性通过质谱法证实，其鉴定了超过一百个蛋白质，其中大多数是已知的内膜蛋白，包括其他已知的极性蛋白质。 该方案或者如果通过选择其它亲和靶适当适当地适合于分离其它感兴趣的膜结构域以鉴定蛋白质或脂质组成。
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