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Millipore/17-484 | phospho-IR (β Subunit) (Tyr 1162/1163) STAR ELISA Assay Kit/17-484/96 assays
  • Millipore/17-484 | phospho-IR (β Subunit) (Tyr 1162/1163) STAR ELISA Assay Kit/17-484/96 assays

Millipore/17-484 | phospho-IR (β Subunit) (Tyr 1162/1163) STAR ELISA Assay Kit/17-484/96 assays

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货号: 17-484
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    • Description
      CatalogueNumber17-484
      BrandFamilyUpstate
      TradeName
      • STAR
      • Upstate
      Descriptionphospho-IR(βSubunit)(Tyr1162/1163)STARELISAAssayKit
      AlternateNames
      • InsulinReceptor
      • IR
      BackgroundInformationI.TESTPRINCIPLE
      TheUPSTATE®colorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheIRplateiscoatedwithaspecificmousemonoclonalIR(βsubunit)captureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardsincludedinthekitareincubatedinthemicrowellsallowingIRtobecapturedintheplatewells.Theplateisthenwashedtoremoveanyunboundnon-specificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-phospho-IRantibodytodetectthecapturedIRphosphorylatedonTyrosines1162and1163.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.
      Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.Thekitalsoincludesastandardthatisrunasbothapositivecontrolandtodevelopastandardcurve.

      II.IRBACKGROUND
      TheInsulinReceptor(IR)issynthesizedasasinglepolypeptide,whichissubsequentlycleavedtogenerateanextracellularαchainandatransmembraneandintracellularβchain,tetheredtogetherbydisulfidebonds.Theβchainhasmultipletyrosinephosphorylationsites,includingthreeautophosphorylationsitesatitsactivationloop.Insulinstimulatesthesignalingcascadebybindingtotheinsulinreceptorthroughitsextracellularαsubunits.Thisstimulatesthetyrosinekinaseactivityoftheβsubunitsofthereceptor.ThereceptorthenautophosphorylatesitselfandphosphorylatestheIRS(InsulinReceptorSubstrate)proteinsthatareveryimportantmodulatorsofinsulinsignaling.PhosphorylatedIRS,inturn,interactswithseveraldownstreameffectorsofinsulinsignaling,includingPI3Kinasep85,GRB2,SHP2,Nck,Crk,andFyn.TheoverallstructureoftheIRishighlyhomologoustotheIGF-IReceptor,exceptintheirc-termini,wherethetwoproteinsdivergesomewhat.InsulinsignalingishighlydependentonthePI3KinasepathwayandAkt,whichappeartomediatethefunctionsofinsulin.
      MaterialsRequiredbutNotDelivered1.Multi-channelorrepeatingPipettes
      2.Plateshaker(optional)
      3.Pipettors&tipscapableofaccuratelymeasuring1-1000µL
      4.GraduatedSEROlogicalpipettes
      5.96-wellmicrotiterPlateReaderwith450nmfilter
      6.Graphingsoftwareforplottingdataorgraphpaperformanualplottingofdata
      7.Microfugetubesforstandardandsampledilutions
      8.Mechanicalvortex
      9.1litercontainer
      10.Distilledordeionizedwater
      ProductInformation
      Components
      • CapturePlatepre-coatedwithanti-IRantibody:(PartNo.17-483A)Onepre-coated96-stripwellimmunoplatesealedinafoilpouch.
      • Anti-phospho-IR(Tyr1162/1163)detectionantibody:(PartNo.17-483B)Onebottle(11mL)ofanti-phospho-IR(Tyr1162/1163)detectionantibodycontainingsodiumazide,readytouse.
      • ELISADiluent:(PartNo.17-483C)Onebottle(25mL)ofELISADiluentcontainingsodiumazide,readytouse.
      • 25XELISAWashBuffer:(PartNo.17-483D)Onebottle(50mL)of25XELISAWashBuffer.
      • Anti-RabbitIgGHRPconjugate:(PartNo.17-483E)Onevial(125µL)of100Xanti-rabbitHRPconjugatecontainingthimerosal.
      • HRPDiluent:(PartNo.17-483F)Onebottle(25mL)ofHRPDiluentcontainingthimerosal.
      • TMBSolution:(PartNo.17-483G)Onebottle(25mL)ofstABIlizedtetramethylbenzidine(TMB),readytouse.
      • StopSolution:(PartNo.17-483H)Onebottle(25mL)ofstopsolution,readytouse.
      • phospho-IRStandard:(PartNo.17-483I)TwovialsofIR((Tyr1162/1163)standard,lyophilized.
      • PlateCovers:Twoplatecovers.
      DetectionmethodChromogenic
      StorageandShippingInformation
      StorageConditionsMaintaintheunopenedkitat2-8°Cuntilexpirationdate.
      Applications
      ApplicationThisphospho-IR(βSubunit)(Tyr1162/1163)STARELISAAssayKitisusedtomeasure&quantifyInsulinlevels.
      KeyApplications
      • ELISA
      BIOLOGicalInformation
      SpeciesReactivity
      • Human
      • Mouse
      • Rat
      AnalytesAvailable
      • Insulin
      EntrezGeneNumber
      EntrezGeneSummaryAfterremovaloftheprecursorsignalpeptide,theinsulinreceptorprecursorispost-translationallycleavedintotwochains(alphaandbeta)thatarecovalentlylinked.Bindingofinsulintotheinsulinreceptor(INSR)stimulatesglucoseuptake.Twotranscriptvariantsencodingdifferentisoformshavebeenfoundforthisgene.
      GeneSymbol
      • INSR
      • CD220
      • IR
      • HHF5
      Modifications
      • Phosphorylation
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:P06213#Thisreceptorbindsinsulinandhasatyrosine-proteinkinaseactivity.IsoformShorthasahigheraffinityforinsulin.Mediatesthemetabolicfunctionsofinsulin.BindingtoinsulinstimulatesassociationofthereceptorwithdownstreammediatorsincludingIRS1andphosphatidylinositol3"-kinase(PI3K).CanactivatePI3Keitherdirectlybybindingtothep85regulatorysubunit,orindirectlyviaIRS1.
      SIZE:1382aminoacids;156307Da
      SUBUNIT:Tetramerof2alphaand2betachainslinkedbydisulfidebonds.Thealphachainscontributetotheformationoftheligand-bindingdomain,whilethebetachainscarrythekinasedomain.InteractswithSORBS1butdissociatesfromitfollowinginsulinstimulation.BindsSH2B2.InteractswiththePTB/PIDdomainsofIRS1andSHC1invitrowhenautophosphorylatedontyrosineresidues.ThesequencessurroundingthephosphorylatedNPXYmotifcontributedifferentiallytoeitherIRS1orSHC1recognition.InteractswiththeSH2domainsofthe85kDaregulatorysubunitofPI3K(PIK3R1)invitro,whenautophosphorylatedontyrosineresidues.InteractswithSOCS7.
      SUBCELLULARLOCATION:Membrane;Single-passtypeImembraneprotein.
      TISSUESPECIFICITY:IsoformLongandisoformShortareexpressedintheperipheralnerve,kidney,liver,striatedmuscle,fibroblastsandskin.IsoformShortisexpressedalsointhespleenandlymphoblasts.
      PTM:AfterbeingtransportedfromtheendoplasmicreticulumtotheGolgiapparatus,thesingleglycosylatedprecursorisfurtherglycosylatedandthencleaved,followedbyitstransporttotheplasmamembrane.&Autophosphorylatedontyrosineresiduesinresponsetoinsulin.&PhosphorylationofTyr-999isrequiredforIRS1-andSHC1-binding.
      DISEASE:SwissProt:P06213#DefectsinINSRarethecauseofinsulinresistance(Insresistance)[MIM:125853].&DefectsinINSRarethecauseofRabson-Mendenhallsyndrome[MIM:262190];alsoknownasMendenhallsyndrome.Itisasevereinsulinresistancesyndromecharacterizedbyinsulin-resistantdiabetesmellituswithpinealhyperplasiaandsomaticabnormalities.Typicalfeaturesincludecoarse,senile-appearingfacies,dentalandskinabnormalities,aBDominaldistension,andphallicenlargement.Inheritanceisautosomalrecessive.&DefectsinINSRarethecauseofleprechaunism[MIM:246200];alsoknownasDonohuesyndrome.Leprechaunismrepresentsthemostsevereformofinsulinresistancesyndrome,characterizedbyintrauterineandpostnatalgrowthretardationanddeathinearlyinfancy.Inheritanceisautosomalrecessive.&DefectsinINSRmaybeassociatedwithnoninsulin-dependentdiabetesmellitus(NIDDM)[MIM:125853];alsoknownasdiabetesmellitustype2.&DefectsinINSRarethecauseoffamilialhyperinsulinemichypoglycemia5(HHF5)[MIM:609968].Familialhyperinsulinemichypoglycemia[MIM:256450],alsoreferredtoascongenitalhyperinsulinism,nesidioblastosis,orpersistenthyperinsulinemichypoglycemiaofinfancy(PPHI),isthemostcommoncauseofpersistenthypoglycemiaininfancyandisduetodefectivenegativefeedbackregulationofinsulinsecretionbylowglucoselevels.&DefectsinINSRarethecauseofinsulin-resistantdiabetesmellituswithacanthosisnigricanstypeA(IRANtypeA)[MIM:610549].Thissyndromeischaracterizedbytheassociationofsevereinsulinresistance(manifestedbymarkedhyperinsulinemiaandafailuretorespondtoexogenousinsulin)withtheskinlesionacanthosisnigricansandovarianhyperandrogenisminadolescentfemalesubjects.Womenfrequentlypresentwithhirsutism,acne,amenorrheaoroligomenorrhea,andvirilization.ThissyndromeisdifferentfromthetypeBthathasbeendemonstratedtobesecondarytothepresenceofcirculatingautoantibodiesagainsttheinsulinreceptor.
      SIMILARITY:SwissProt:P06213##Belongstotheproteinkinasesuperfamily.Tyrproteinkinasefamily.Insulinreceptorsubfamily.&Contains2fibronectintype-IIIdomains.&Contains1proteinkinasedomain.
      PhysicochemicalInformation
      Sensitivity
      • Sensitivity:1Unit/mL
        RangeofDetection:1.6-100Units/mL
      Dimensions
      MaterialsInformation
      MaterialsInformation
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