Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
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DOI：
10.3791/3916
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发表时间：
2012-05-01
影响因子：
1.2
通讯作者：
Goldberg, Joanna B.
中科院分区：
文献类型：
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作者：
Davis, Michael R.;Goldberg, Joanna B.
脂多糖（LPS）是革兰氏阴性菌外膜的主要成分。它是一种三重分子，由包埋在外膜中的脂质A、核心寡糖和从细胞表面向外延伸的重复O-抗原单元组成（1，2）。LPS是一种免疫显性分子，对许多细菌物种的毒力和致病性都很重要，包括铜绿假单胞菌、沙门氏菌和大肠杆菌（3-5），LPS O-抗原组成的差异构成了菌株血清分型的基础。LPS在感染开始时参与与宿主细胞的附着，并提供对补体介导的杀伤的保护;缺乏LPS的菌株可以减毒（6-8）。由于这些原因，重要的是可视化LPS，特别是来自临床分离株的LPS。可视化的LPS带型和识别特异性抗体可以是有用的工具，以确定菌株谱系和表征各种mutants.In这份报告中，我们描述了一种热水酚法从革兰氏阴性菌细胞的LPS的分离和纯化。该方案允许从核酸和蛋白质中提取LPS，这些核酸和蛋白质可能干扰LPS的可视化，而LPS的可视化是在较短、强度较低的提取方法中发生的（9）。以这种方式制备的LPS可以通过十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）分离，并使用碳水化合物/糖蛋白染色剂或标准银染色方法直接染色。许多针对LPS的抗血清含有与外膜蛋白或其他抗原靶标交叉反应的抗体，所述抗体可阻碍在SDS-PAGE分离的粗细胞裂解物的Western免疫印迹后观察到的反应性。单独的粗细胞裂解物的蛋白酶处理并不总是使用这种或其他可视化方法去除该背景的有效方式。此外，试图去除该背景的大量蛋白酶处理可导致通过任何上述方法不能很好地分辨的质量差的LPS。出于这些原因，我们认为以下方案（改编自Westpahl和Jann（10））是LPS提取的理想方案。
Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell(1,2). LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli(3-5), and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence(6-8). For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants.In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods(9). LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann(10), is ideal for LPS extraction.