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CatalogueNumber | 17-483 |
BrandFamily | Chemicon® |
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Description | InsulinReceptor(βsubunit)STARELISAAssayKit |
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BackgroundInformation | I.TESTPRINCIPLE TheUPSTATE®colorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheIRplateiscoatedwithaspecificmousemonoclonalIR(βsubunit)captureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardsincludedinthekitareincubatedinthemicrowellsallowingIRtobecapturedintheplatewells.Theplateisthenwashedtoremoveanyunboundnon-specificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-IRantibodytodetectthecapturedIR(βsubunit)ontheplatewell.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-channelorrepeatingPipettes2.Plateshaker(optional)3.Pipettors&tipscapableofaccuratelymeasuring1-1000µL4.GraduatedSEROlogicalpipettes5.96-wellmicrotiterPlateReaderwith450nmfilter6.Graphingsoftwareforplottingdataorgraphpaperformanualplottingofdata7.Microfugetubesforstandardandsampledilutions8.Mechanicalvortex9.1litercontainer10.Distilledordeionizedwater |
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Detectionmethod | Chromogenic |
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StorageConditions | Maintaintheunopenedkitat2-8°Cuntilexpirationdate. Precautions •Theinstructionsprovidedhavebeendesignedtooptimizethekit"sperformance.Deviationfromtheinstructionsmayresultinsuboptimalperformanceofthekitandthefailuretoproduceaccuratedata. •CausticMaterial:StopSolution.Caution:Eye,hand,face,andclothingprotectionshouldbewornwhenhandlingthismaterial. •SafetyWarningsandPrecautions:Thiskitisdesignedforresearchuseonlyandnotrecommendedforinternaluseinhumansoranimals.Allchemicalsshouldbeconsideredpotentiallyhazardousandprinciplesofgoodlaboratorypracticeshouldbefollowed. •TheDetectionAntibodyandELISADiluentcontainsodiumazide.Sodiumazidemayreactwithcopperandleadplumbingtoformhighlyexplosivemetalazides.Upondisposal,flushwithlargeamountsofwatertopreventazidebuild-up.Avoidcontactwithskin. •TheAnti-RabbitIgGHRPConjugateandHRPDiluentcontainthimerosal.Thimerosalishighlytoxicbyinhalation,contactwithskinorifswallowed.ThimerosalisapossIBLemutagenandshouldbehandledaccordingly. |
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Application | ThisInsulinReceptor(βsubunit)STARELISAAssayKitisusedtomeasure&quantifyInsulinlevels. |
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BIOLOGicalInformation | |
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EntrezGeneNumber | |
EntrezGeneSummary | Afterremovaloftheprecursorsignalpeptide,theinsulinreceptorprecursorispost-translationallycleavedintotwochains(alphaandbeta)thatarecovalentlylinked.Bindingofinsulintotheinsulinreceptor(INSR)stimulatesglucoseuptake.Twotranscriptvariantsencodingdifferentisoformshavebeenfoundforthisgene. |
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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. |
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