Name: purification and visualization of lipopolysaccharide from gram-negative bacteria by hot aqueous-phenol extraction
Uploaded: 2012-05-28T12:00:00
Description: Watch this Scientific Journal Video about Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction at JoVE.com

Dette arbejde blev støttet af tilskud fra National Institutes of Health og cystisk fibrose Foundation.

1. Fremstillinq af bakterier til LPS Ekstraktion <ol><li> Begynder en overnatskultur i 5 ml Luria Broth (LB), suppleret med antibiotika, hvis nødvendigt. Vokser kultur natten over (12-18 timer) i en rysteinkubator ved 37 ° C og 200 rpm. </li><li> Fortynd kulturen 1:10 med LB og tage en OD600 aflæsning i et spektrofotometer. Baseret på OD600 aflæsning, en 1,5 ml suspension af Deres bakterier gør en OD600 på 0,5. </li><li> Pellets af bakterier i en mikrocentrifuge ved 10.600 x <em...

<ol>
	<li>Raetz, C. R., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+endotoxins.">Lipopolysaccharide endotoxins.</a> Annu. Rev. Biochem. 71, 635-700 (2002).</li><li>Valvano, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Export+of+O-specific+lipopolysaccharide.">Export of O-specific lipopolysaccharide.</a> Front. Biosci. 8, 452-471 (2003).</li><li>Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide:+a+major+virulence+factor,+initiator+of+inflammation+and+target+for+effective+immunity.">Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity.</a> Int. J. Med. Microbiol. 297, 277-295 (2007).</li><li>Freudenberg, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipopolysaccharide+susceptibility+in+the+innate+immune+response+to+Salmonella+typhimurium+infection:+LPS,+a+primary+target+for+recognition+of+Gram-negative+bacteria.">Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.</a> Microbes Infect. 3, 1213-1222 (2001).</li><li>Jann, K., Jann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+antigens+of+Escherichia+coli.">Polysaccharide antigens of Escherichia coli.</a> Rev. Infect. Dis. 9, S517-S526 (1987).</li><li>McCallum, K. L., Schoenhals, G., Laakso, D., Clarke, B., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-molecular-weight+fraction+of+smooth+lipopolysaccharide+in+Klebsiella+serotype+O1:K20+contains+a+unique+O-antigen+epitope+and+determines+resistance+to+nonspecific+serum+killing.">A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.</a> Infect. Immun. 57, 3816-3822 (1989).</li><li>Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+isolates+from+patients+with+cystic+fibrosis:+a+class+of+serum-sensitive,+nontypable+strains+deficient+in+lipopolysaccharide+O+side+chains.">Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains.</a> Infect. Immun. 42, 170-177 (1983).</li><li>Gupta, S. K., Masinick, S., Garrett, M., Hazlett, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide+binds+galectin-3+and+other+human+corneal+epithelial+proteins.">Pseudomonas aeruginosa lipopolysaccharide binds galectin-3 and other human corneal epithelial proteins.</a> Infect. Immun. 65, 2747-2753 (1997).</li><li>Hitchcock, P. J., Brown, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+heterogeneity+among+Salmonella+lipopolysaccharide+chemotypes+in+silver-stained+polyacrylamide+gels.">Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.</a> J. Bacteriol. 154, 269-277 (1983).</li><li>Westphal, O., Jann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lipopolysaccharides.">Bacterial lipopolysaccharides.</a> Methods Carbohyr. Chem. 5, 83 (1965).</li><li>Lieberman, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+bacterial+evolution+within+multiple+patients+ties+novel+genes+to+pathogenesis.">Parallel bacterial evolution within multiple patients ties novel genes to pathogenesis.</a> Nat Genet. , (2011).</li></ol>

Vi har beskrevet en fremgangsmåde til oprensning af LPS fra andre cellulære bestanddele, herunder nukleinsyrer og proteiner. Denne fremgangsmåde tilvejebringer høj kvalitet LPS, som kan anvendes i en række forskellige visualisering metoder, herunder carbohydrat farvning af SDS-PAGE-geler, som vist i figur 1. Denne fremgangsmåde kan anvendes til serotype LPS fra forskellige stammer ved anvendelse af specifikke anti-sera, eller at vise slægtskab mellem isolater ved direkte visualisering. For eksempel fra en nylig g...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> DNase I rekombinant, RNase-fri </td><td> Roche </td><td> 04716728001 </td></tr><tr><td> RNase A </td><td> Roche </td><td> 10109169001 </td></tr><tr><td> Proteinase K </td><td> Fisher </td><td> BP1700 </td></tr><tr><td> Tris-mættet phenol </td><td> Fisher </td><td> BP1750-100 </td></tr><tr><td> Diethylether </td><td> Thomas Scientific </td><td> C313K31 </td></tr><tr><td> Pro-Q Emerald 300 Lipopolysaccharid Gel Stain Kit </td><td> Molecular Probes </td><td> P20495 </td></tr></tbody></table>

purification and visualization of lipopolysaccharide from gram-negative bacteria by hot aqueous-phenol extraction

Lipopolysaccharid (LPS) er en vigtig bestanddel af Gram-negative bakterielle ydre membraner. Det er et tredelt molekyle bestående af lipid A, der er indlejret i den ydre membran, en kerne oligosaccharid og gentagelse O-antigen-enheder, som strækker sig udad fra overfladen af cellen 1, 2. LPS er en immundominant molekyle, som er vigtig for virulens og patogenesen af mange bakteriearter, herunder Pseudomonas aeruginosa, Salmonella-arter, og Escherichia coli 3-5, og forskelle i LPS O antigen-præparat danner grundlaget for serotypning af stammer. LPS er involveret i binding til værtsceller ved initiering af infektionen og giver beskyttelse af komplement-medieret drab; stammer, som mangler LPS kan dæmpes for virulens 6-8. Af disse grunde er det vigtigt at visualisere LPS, især fra kliniske isolater. Visualisering LPS båndmønstre og anerkendelse af specifik enntibodies kan være nyttige værktøjer til at identificere stamme slægter og at karakterisere de forskellige mutanter. I denne rapport beskriver vi en varm vandig phenol fremgangsmåde til isolering og oprensning af LPS fra Gram-negative bakterielle celler. Denne protokol muliggør ekstraktion af LPS væk fra nukleinsyrer og proteiner, der kan interferere med visualisering af LPS, der forekommer med kortere og mindre intensive ekstraktionsmetoder 9. LPS fremstillet på denne måde kan adskilles ved natriumdodecylsulfat (SDS)-polyacrylamidgelelektroforese (PAGE) og direkte farvet med carbohydrat / glycoprotein pletter eller faste sølvfarvning metoder. Mange anti-sera til LPS indeholde antistoffer, som krydsreagerer med ydre membran-proteiner eller andre antigene mål, som kan hæmme reaktivitet blev observeret efter Western immunoblot af SDS-PAGE-separerede rå cellelysater. Proteasebehandling af rå cellelysater alene ikke altid en effektiv måde at fjerne dennebaggrund ved hjælp af denne eller andre visualisering metoder. Yderligere kan store proteasebehandling i et forsøg på at fjerne denne baggrund føre til dårlig kvalitet LPS, som ikke kan løses ved enhver af de førnævnte fremgangsmåder. Af disse grunde mener vi, at følgende protokol, tilpasset fra Westpahl og Jann 10, er ideel til LPS ekstraktion.

Watch this Scientific Journal Video about Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction at JoVE.com

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Vi beskriver en modificeret varm vandig ekstraktion med phenol fremgangsmåde til oprensning af lipopolysaccharid (LPS) fra gramnegative bakterier. Når først ekstraheres, kan de LPS efterfølgende analyseret ved SDS-PAGE og visualiseret ved direkte farvning eller Western immunoblot.

Oprensning og visualisering af lipopolysaccharid fra Gram-negative bakterier ved varm vandig-phenolekstraktion

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Research

JoVE Journal

Immunology and Infection

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex protokol til DNA-ekstraktion fra<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Oprensning af Patogen vakuoler fra<em&gt; Legionella</em&gt;-Inficerede Fagocytter

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

En fremgangsmåde til frembringelse Pulmonal Neutrofili Brug Aerosolized Lipopolysaccharid

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

Rapid Identifikation af Gram-negative bakterier fra Blood kulturvæsken Brug MALDI-TOF-massespektrometri

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

T-celler Capture bakterier ved Transinfection fra dendritiske celler

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Bestemmelse af biofilm Initiation på virusinficerede celler af bakterier og svampe

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Isolation, karakterisering og rensning af makrofager fra væv ramt af fedme-relaterede Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Ekstraktion af Hemocytes fra Drosophila melanogaster larver for mikrobiel infektion og analyse

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...