ग्राम नकारात्मक जीवाणुओं से शोधन और गरम निकालना जलीय - फिनोल द्वारा Lipopolysaccharide का विजुअलाइज़ेशन
Summary
हम एक संशोधित गर्म जलीय phenol के ग्राम नकारात्मक जीवाणुओं से lipopolysaccharide (LPS) सफ़ाई के लिए निष्कर्षण विधि का वर्णन. एक बार निकाले LPS बाद एसडीएस पृष्ठ से विश्लेषण किया जा सकता है और प्रत्यक्ष धुंधला हो जाना या पश्चिमी immunoblot के द्वारा कल्पना.
Abstract
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 cell1, 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 coli3-5, 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 virulence6-8. 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 methods9. 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 Jann10, is ideal for LPS extraction.
Protocol
Discussion
हम LPS न्यूक्लिक एसिड और प्रोटीन सहित अन्य सेलुलर घटक, से दूर शुद्ध करने की एक विधि का वर्णन किया है. इस विधि उच्च गुणवत्ता LPS कि एसडीएस पृष्ठ जैल की कार्बोहाइड्रेट धुंधला सहित विभिन्न दृश्य तरीकों, जैसा क?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम के स्वास्थ्य और सिस्टिक फाइब्रोसिस फाउंडेशन के राष्ट्रीय संस्थानों से अनुदान द्वारा समर्थित किया गया.
Materials
| Name of the reagent | Company | Catalogue number |
| DNase I recombinant, RNase-free | Roche | 04716728001 |
| RNase A | Roche | 10109169001 |
| Proteinase K | Fisher | BP1700 |
| Tris-Saturated Phenol | Fisher | BP1750-100 |
| Diethyl Ether | Thomas Scientific | C313K31 |
| Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit | Molecular Probes | P20495 |
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