Description
CatalogueNumber	17-484
BrandFamily	Upstate
TradeName	STAR Upstate
Description	phospho-IR(βSubunit)(Tyr1162/1163)STARELISAAssayKit
AlternateNames	InsulinReceptor IR
BackgroundInformation	I.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.
MaterialsRequiredbutNotDelivered	1.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.
Detectionmethod	Chromogenic

StorageandShippingInformation
StorageConditions	Maintaintheunopenedkitat2-8°Cuntilexpirationdate.

Applications
Application	Thisphospho-IR(βSubunit)(Tyr1162/1163)STARELISAAssayKitisusedtomeasure&quantifyInsulinlevels.
KeyApplications	ELISA

BIOLOGicalInformation
SpeciesReactivity	Human Mouse Rat
AnalytesAvailable	Insulin
EntrezGeneNumber	NM_000208.2 NM_001079817.1
EntrezGeneSummary	Afterremovaloftheprecursorsignalpeptide,theinsulinreceptorprecursorispost-translationallycleavedintotwochains(alphaandbeta)thatarecovalentlylinked.Bindingofinsulintotheinsulinreceptor(INSR)stimulatesglucoseuptake.Twotranscriptvariantsencodingdifferentisoformshavebeenfoundforthisgene.
GeneSymbol	INSR CD220 IR HHF5
Modifications	Phosphorylation
UniProtNumber	P06213
UniProtSummary	FUNCTION: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

Millipore 提供三种不同的 Immobilon PVDF 膜，是针对不同的蛋白质印迹应用的理想选择。 现在我们还 提供采用按规格裁切的印迹方片膜和两层过滤纸组成的印迹三明治。   与硝酸纤维素膜相比，Immobilon 膜具有多种优点。 它们不易破裂或卷 曲，便于裁剪且在切割过程中不会碎裂。 它们还有低背景、广泛溶剂相容性以及优异的着色能力。 此外，它们可以重复检测。 Immobilon-P 膜（沃比森-供应） ·         0.45μm 孔径 ·         建议用于大多数免疫印迹，特别在蛋 白质大于 20kDa 时 ·         Millipore 的快速免疫检测实验方法不需要膜封闭并缩短洗涤时间，将检测时间至少减少了两个小时，而不致降低灵敏 度或特异性 用于高通量实验室的印迹三明治 ·         当您需要处理多个印迹时，印迹三明治是一种便利省时的选择。 我们的印迹三明 治包含多片 Immobilon-P 膜，其中插入了按规格裁切的多张层析级印迹纸。 转印滤纸 ·         按照大多数免疫印迹尺寸进行裁切的层析级转印滤纸。 Immobilon-PSQ 膜（沃比森-供应） ·         0.2μm 孔径和大的内表面面积 ·         较其它膜具有更高的 蛋白质吸附率和测序得率 ·         建议用于小于 20kDa 的印迹蛋白质 ·         防止小分子量的蛋白质的渗漏 Immobilon-FL 膜（沃比森-供应） ·        针对荧光应用进行优化的转印膜 ·         极低的背景提高了所 有荧光检测实验的灵敏度 ·         与所有常用的荧光探针（所有激发和发射波长）兼容 ·         是多态性检测和化学荧光应用的理想选择