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LipopolysaccharidesfromEscherichiacoli055:B5ProductNumberL2880StorageTemperature2-8°CProductDescriptionSynonym:LPSThisproductisphenolextractedfromE.coliserotypeO55:B5.ThesourcestrainisCDC1644-70.TheLPSO55:B5hasbeenusedtostimulatehumanperitonealmacrophagesat1ng/ml22andtostimulateequineperitonealmacrophagesat1-100ng/ml23.Lipopolysaccharides(LPS)arecharacteristiccomponentsofthecellwallofGramnegativebacteria;theyarenotfoundinGrampositivebacteria.Theyarelocalizedintheouterlayerofthemembraneandare,innoncapsulatedstrains,exposedonthecellsurface.Theycontributetotheintegrityoftheoutermembrane,andprotectthecellagainsttheactionofbilesaltsandlipophilicantibiotics.1Lipopolysaccharidesaremadeupofahydrophobiclipid(lipidA,whichisresponsibleforthetoxicpropertiesofthemolecule),ahydrophiliccorepolysaccharidechain,andahydrophilicO-antigenicpolysaccharidesidechain.Inmostcases,O-specificchainsarebuiltofrepeatingunitsofoligosaccharideswhichexhibitastrain-specificstructuraldiversity.Thesugarconstituents,theirsequence,andtheirmodeoflinkagedeterminetheserologicalOspecificityofrespectivestrains.TheyarethemaindeterminantsoftheclassificationsoftheserotypesofSalmonellaspecies.ThediversityofOchainsinEnterobacteriaceaemayhavedevelopedduringevolutiontoallowentericbacterialtoescapethehost’simmunesystembydevelopingnewspecificitiesontheircellsurface(specifictothebacterialserotype).1Sincelipopolysaccharidesconferantigenicpropertiesonthecell,theyhavebeentermedOantigens.Asthemainantigen,lipopolysaccharidesareinvolvedinvarioushost-parasiteinteractions.TheyseemtoprotectGramnegativebacteriafromphagocytosisandlysis.1Bacteriawithcommonserotypeshavesurfaceantigens(groupO,groupH,orLPS)whichgeneratethesameantibodyresponse.ExamplesofserotypesareO55:B5andO26:B6fortheE.colibacterium.Thedesignationsareimmunologicalclassifications,whichspecifywhichantibodyrecognizedwhichstrains.Differentstrainsmayhavesomecommonantigenicdeterminants.IfawildstrainofbacteriumisirradiatedwithUVlightorexposedtomutageniccompounds,itwillmutate.Thefewmutationsthatarenotlethalresultinviablemutants(roughstrains)whicharegenerallynotfoundinnature,andwhichpossesssomeuniquecharacteristics.Thegenesthatencodelipopolysaccharideformationmayalsobealteredinthemutants,andLPSwithshorterpolysaccharidechainsmaybeformed.Ra,Rb,Rc,Rd,Re,etc.(wherea,b,c,etc...designate1st,2nd,3rd,etc...degree,respectively)designatethepolysaccharidelengthofagivenLPS.RaandRedesignatethemutantswiththelongestandshortestchainlengths,respectively.2ThemostextrememutantsaretheRemutantswhichproduceanLPSwhichismadeupofLipidAand3-deoxy-D-manno-octulosonicacid(2-Keto-3-deoxyoctonate,KDO)asthesoleconstituentofthecore.2LipidAandlipopolysaccharidesfromroughstrainsaretestedforKDOcontent.3PurifiedendotoxinisgenerallyreferredtoaslipopolysaccharideorLPS,todistinguishitfromthemorenaturalcomplexedcellmembraneassociatedform.ThecoreportionofthepolysaccharidechainiscommontoLPSfromwildandmutantbacterialstrains.RemovalbyhydrolysisofthepolysaccharidechainfromLPSproduceslipidA,eitherasthenaturallyoccurring,cytotoxicdiphosphorylform4orthelesstoxic,monophosphorylform.5,6Thelongerthepolysaccharidechainis,thelongerandmoredifficultthehydrolysis.LPSwithalongpolysaccharidechainhasarelativelylowlipidAcontent,whichmustbepurifiedfromalargeamountofhydrolysisbyproducts(oligosaccharidesandsaccharidemonomers).Thus,theyieldoflipidAislowandrecoveryispoor.LPSwithashortpolysaccharidechain(LPSfrommutantbacteria)isthereforeusedtoproducelipidAproducts.RemovalofthefattyacidportionsoflipidAresultsinadetoxifiedLPS7withanendotoxinlevelabout10,000timeslowerthanthatoftheparentLPS.ThemolecularstructureofLPShasbeenstudied.8,9SinceLPSisheterogeneousandtendstoformaggregatesofvaryingsizes,themolecularweightisnotverymeaningful.However,thereisareportedrangeof1-4millionorgreater.WhentheLPSistreatedwithSDSandheat,themolecularweightisintherangeof50to100kDa.Intheirpurestform,inthepresenceofstrongsurfaceactiveagents,andintheabsenceofdivalentcations,bacterialendotoxinsconsistof10-20kDamacromolecules.IntheabsenceofsurfaceactiveagentsandinthepresenceofdivalentcationsequesteringagentssuchasEDTA,LPSisbelievedtoarrangeitselfintoamicellarstructurewithamolecularweightofapproximately1,000kDa.ThisisthesmallestformofbacterialLPSthatislikelytoexistinaqueousliquids.InthepresenceofdivalentcationssuchasCa2+andMg2+,abilayerstructureappearstoexistthatpassesthrougha0.2µmmembrane,butdoesnotpassthrougha0.025µmmembrane.LPSvesiclesupto0.1µmindiametermayalsobeformedinwaterinthepresenceofdivalentcations.TheselfaggregationofLPSisgenerallyafunctionofthelipidAcomponentofthemolecule,whichalsoconferstheabilitytobindtohydrophobicsurfaces.LPScanbepreparedbyTCA,10phenol,11orphenol-chloroform-petroleumether(forroughstrains)12extraction.TheTCAextractedlipopolysaccharidesarestructurallysimilartothephenolextractedones.Theirelectrophoreticpatternandendotoxicityaresimilar.Themaindifferencesareintheamountsofnucleicacidandproteincontaminations.TheTCAextractcontainsapproximately2%RNAandapproximately10%denaturedproteins.Theph
本文标题:LPS(sigma)说明书
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