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Millipore/NS170 | Phosphorylated Neurofilament (pNF-H) Sandwich ELISA Kit/NS170/1 kit96 wells
  • Millipore/NS170 | Phosphorylated Neurofilament (pNF-H) Sandwich ELISA Kit/NS170/1 kit96 wells

Millipore/NS170 | Phosphorylated Neurofilament (pNF-H) Sandwich ELISA Kit/NS170/1 kit96 wells

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货号: NS170
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    • Description
      CatalogueNumberNS170
      BrandFamilyChemicon®
      TradeName
      • Chemicon
      DescriptionPhosphorylatedNeurofilament(pNF-H)SandwichELISAKit
      BackgroundInformationTheChemiconPhosphorylatedNeurofilamentH(pNF-H)kitisasandwichenzymeimmunoassay(EIA),whichmeasurespNF-H.ThekitwillmeasurepNF-Hfromavarietyofmammalianspeciesincludinghuman,rat,mouse,bovine,porcine,andfeline.

      Neurofilamentsarethe10nmdiameterfilamentswhichmakeupthemajorpartoftheCytoskeletonofneuronsandareparticularlyconcentratedinlargerdiameteraxons.Theybelongtotheintermediateor10nmfilamentprotein/genesuperfamilywhichalsoincludesthekeratins,themajorstructuralproteinsofskin.NeurofilamentsaregenerallyregardedasbeingcomposedofthreemajorproteinsubunitsreferredtoasNF-L,NF-MandNF-H.NF-Listhe"low"or"light"subunit,NF-Misthe"medium"or"middle"subunitandNF-Histhe"heavy"or"high"subunit.ThisnomenclatureisbasedontheapparentsizeoftheseproteinsonSDS-PAGEgels;NF-Listypicallyabout68kDa,NF-Misabout150kDaandNF-Hisabout200kDa.Thesethreeproteins,referredtoastheneurofilamenttriplet,werediscoveredinthe1970sasaresultofstudiesofaxonallytransportedproteins(HoffmanandLasek1975).Afourthprotein,α-internexin,a.k.aNF66,waslaterdiscoveredasaproteinco-purifiingwiththeneurofilamenttripletproteins(PachterandLiem1985).Afifthprotein,peripherin,isfoundinsomeneurofilamentsalongwiththeotherfourproteins,mostlyintheperipheralnervoussystem(Portieretal.1983).Neurofilamentsaremajorproteinsofneuronsandareparticularlyconcentratedinaxons.TheproteinNF-Hhassomeveryunusualproperties.Firstlyitcontains50-60backtobackhexa,heptaoroctapeptiderepeatseachcontainingthesequenceLysine-Serine-Proline(KSP).Theserineresiduesinthesepeptiderepeatsarephosporylatedinvivo,andinaxonalneurofilaments,thesesitesareheavilyphosphorylated.ThephosphorylatedformsofNF-Harealsoquiteresistanttoproteases,suggestingthatthisveryabundantproteinmightbeparticularlyresistanttoproteotyticcleavageuponreleasefromdamagedanddiseasedaxons.Thismeansthatdetectionofthisproteinincerebrospinalfluid(CSF)andbloodwouldprovideinformationaboutthedegreeofaxonalinjurywhichhasoccurred(reviewedinPetzold2005).Asrecentlyshown,thisproteincanbedetectedinquitelargeamountsfollowingexperimentalspinalcordandbraininjuryinrats(Shawetal.2005).Levelsofgreaterthan100ng/mlofpNF-Hweredetectableinbloodfollowingseriousspinalcordinjuryandlowerbutstilleasilydetectablelevelswereseeninbloodofanimalsgivenexperimentalbraininjury.Morerecentstudieshaverevealedconsiderableamountsofthisproteininthebloodofmicetransgenicformutationsofhumancopper/zincsuperoxidedismutase1whichareassociatedwithamyotrophiclateralsclerosis(ALS).ThesemicedevelopanaxonaldegenerationpathologysimilartothatseeninhumanswithALS,andbloodpNF-Hlevelscanbeusedtomonitorthis.Interestingly,pNF-Hisdetectablebeforetheonsetofobviousdiseasesymptoms.OtherstudiesshowthatpNF-Hcanbedetectedintissuehomogenates.OtherexperimentsshowthatpNF-Hcanbedetectedintheplasmaofhumanssufferingfromopticneuritis(Petzoldetal2004)andinelevatedlevelsinthecerebrospinalfluid(CSF)ofindividualssufferingfrombraintumorsandstroke(PetzoldandShaw,unpublished).

      TestPrincipleWiththeChemiconpNF-Hassaysystem,chickenpolyclonalantibodiesgeneratedagainstpNF-H,arepre-coatedontoa96-wellplateandareusedtocapturepNF-Hfromasample.CapturedpNF-HisdetectedusingpNF-Hspecificrabbitpolyclonalantibodiesandagoatanti-rabbitalkalinephosphataseconjugate.AfteradditionofthesubstratesolutiontheamountofpNF-Hisdetermined.ThestandardcurvedemonstratesadirectrelationshipbetweenOpticalDensity(OD)andpNF-Hconcentration:i.e.,thehighertheODthehigherthepNF-Hconcentrationinthesample.

      ApplicationTheChemiconpNF-HkitisdesignedtomeasuretheamountofpNF-HintissuehomogenatesandBIOLOGicalfluid(serum,plasma,andserum-free)samplesfrommouseandrat.Thereareenoughreagentsincludedinthiskitforone96-wellimmuno-assayplate.Runningduplicatewellsforsamplesandstandardsisrecommended.
      MaterialsRequiredbutNotDelivered1.Multi-channelorrepeatingPipettes
      2.Pipettors&tipscapableofaccuratelymeasuring10-1000μL
      3.GraduatedSEROlogicalpipettes
      4.96-wellmicroplatereadercapableofmeasuringabsorbanceat405nm
      5.Graphpaperformanualplottingofdata
      6.1.5mLtubes
      7.Mechanicalvortex
      8.Two1litercontainers
      9.Plateshaker(optional)
      10.3NNaOH(optional)
      11.Distilledordeionizedwater.
      ProductInformation
      Components
      • ImmunoplatePre-coatedwithChickenanti-pNF-HPolyclonalAntibody:(PartNo.2006263).One96-wellimmunoplatepre-coatedwithchickenanti-pNF-Hpolyclonalantibodysealedinafoilpouch.
      • Rabbitanti-pNF-HPolyclonalAntibody:(PartNo.2006919).Onevial(100μL)ofrabbitanti-pNF-Hpolyclonalantibody.
      • Goatanti-Rabbit,AlkalinePhosphataseConjugatedAntibody:(PartNo.2006917).Onevial(10μL)ofgoatanti-rabbitalkalinephosphataseconjugate.
      • BovinepNF-HStandard:(PartNo.2006918).Onevial(5.0ng)ofbovinepNF-Hstandard.
      • 20XTBS:(PartNo.2004913).Onebottle(50mL)of20XTBS.
      • 20%Tween-20™:(PartNo.2004915).Onevial(3mL)of20%Tween-20™.
      • ElisaAssayDiluent:(PartNo.2006921)Onebottle(30mL)ofElisaassaydiluentcontaing0.05%Kathonasapreservative,providedinareadytouseformat.
      • pNPPBuffer:(PartNo.2006261).Onebottle(12mL)ofpNPPbufferforusewith50Xp-NPP,providedinareadytouseformat.
      • 50XpNPP:(PartNo.2006262).Onevial(250μL)of50XpNPP(p-nitrophenylphosphatase)alkalinephosphatasesubstrate.
      • PlateCovers-TwoPlateCovers.
      DetectionmethodChromogenic
      StorageandShippingInformation
      StorageConditionsPriortousemaintainthekitcomponentsat2°-8°Cand-20°C,asindicated,untilexpirationdate.

      1.WashBuffer-AfterdilutionmaintaintheWashSolutionat2°-8°Cforuptoonemonthoruntilexpirationdate.

      2.Tween-20™-Storeatroomtemperatureuponreceiptofkit.

      3.ElisaAssayBuffer-Aliquotasrequiredtoavoidrepeatedfreeze-thawcycles.Afterthawing,maintaintheElisaAssayBufferat2°-8°Cforuptoonemonth,oruntilexpirationdate.

      4.ImmunoplatePre-coatedwithChickenanti-pNF-HPolyclonalAntibody-Unusedstripwellsmaybereturnedtothefoilpouchandstoredat2°-8°Cforuptoonemonth,oruntilexpirationdate.
      Applications
      ApplicationThePhosphorylatedNeurofilamentH(pNF-H)kitisasandwichenzymeimmunoassay(EIA),whichmeasurespNF-H.
      KeyApplications
      • ELISA
      BiologicalInformation
      SpeciesReactivity
      • Bovine
      • Human
      • Mouse
      • Pig
      • Rat
      • Feline
      AnalytesAvailable
      • PhosphorylatedNeurofilament(pNF-H)
      EntrezGeneNumber
      GeneSymbol
      • NEFH
      • NF-H
      • NFH
      • KIAA0845
      Modifications
      • Phosphorylation
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:P12036#Neurofilamentsusuallycontainthreeintermediatefilamentproteins:L,M,andHwhichareinvolvedinthemaintenanceofneuronalcaliber.NF-HhasanimportantfunctioninmatureaxonsthatisnotsubservedbythetwosmallerNFproteins.
      SIZE:1026aminoacids;112480Da
      PTM:ThereareanumberofrepeatsofthetripeptideK-S-P,NFHisphosphorylatedonanumberoftheserinesinthismotif.ItisthoughtthatphosphorylationofNFHresultsintheformationofinterfilamentcrossbridgesthatareimportantinthemaintenanceofaxonalcaliber.&Phosphorylationseemstoplayamajorroleinthefunctioningofthelargerneurofilamentpolypeptides(NF-MandNF-H),thelevelsofphosphorylationbeingaltereddevelopmentallyandcoincidentwithachangeintheneurofilamentfunction.
      DISEASE:SwissProt:P12036#DefectsinNEFHareacauseofsusceptibilitytoamyotrophiclateralsclerosis(ALS)[MIM:105400].ALSisaneurodegenerativedisorderaffectingupperandlowermotorneurons,andresultinginfatalparalysis.Sensoryabnormalitiesareabsent.Deathusuallyoccurswithin2to5years.Theetiologyislikelytobemultifactorial,involvingbothgeneticandenvironmentalfactors.
      SIMILARITY:SwissProt:P12036##Belongstotheintermediatefilamentfamily.
      PhysicochemicalInformation
      Sensitivity
      • Sensitivity:0.0585ng/mL
        RangeofDetection:0.0293ng/mLto15ng/mL
      Dimensions
      MaterialsInformation
      MaterialsInformation
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