DescriptionCatalogueNumberHTS001MBrandFamilyChemicon®TradeNameChemiScreenChemiconDescriptionChemiSCREEN™MembranePreparationRecombinantHumanCXCR1ChemokineReceptorOverview:Full-lengthhumanCXCR1CDNABackgroundInformationCXCR1(alsoknownasIL-8RA)isaGi/o-coupledGPCRexpressedonneutrophils,CD8(+)Tcells,andintestinalepithelialcells(Murphy,1997;Sturmetal.,2005;Takataetal.,2004).CXCR1bindsspecificallytothechemokineinterleukin-8(IL-8),whereasarelatedreceptor,CXCR2,bindspromiscuouslytoIL-8andseveralotherchemokines.CXCR1hasbeenproposedtobetheprimaryregulatorofneutrophilresponsetoIL-8insepsis(Cummingsetal.,1999).AntagoNISTsofCXCR1andCXCR2havebeendevelopedanddisplayefficacyinanimalmodelsofreperfusioninjury(Bertini,etal.,2004;Kaneideretal.,2005).Millipore"sCXCR1membranepreparationsarecrudemembranepreparationsmadefromourproprietarystablerecombinantcelllinestoensurehigh-levelofGPCRsurfaceexpression;thus,theyareidealHTStoolsforscreeningofantagonistsofCXCR1interactionwithIL-8.ThemembranepreparationsexhibitaKdof0.2nMfor[125I]-IL-8.With5μg/wellCXCR1MembranePrepand0.1nM[125I]-IL-8,agreaterthan5-foldsignal-to-backgroundratioisobtained.ProductInformationFormatMembranesPresentationLiquidinpackagingbuffer:50mMTrispH7.4,10%glyceroland1%BSAnopreservatives.Packagingmethod:Membranesproteinwereadjustedtotheindicatedconcentrationinpackagingbuffer,rapidlyfrozen,andstoredat-80°C.StorageandShippingInformationStorageConditionsMaintainfrozenat-70°Cforupto2years.Donotfreezeandthaw.ApplicationsApplicationHumanCXCR1/IL-8aGPCRmembranepreparationforRADIoligandbindingAssays>PgammaSbinding.KeyApplicationsRadioligandbindingassayGTPγSbindingApplicationNotesRadioligandbindingassay,andGTPgammaSbinding.BIOLOGicalInformationConcentration1mg/mLEntrezGeneNumberNM_000634.2EntrezGeneSummaryTheproteinencodedbythisgeneisamemberoftheG-protein-coupledreceptorfamily.Thisproteinisareceptorforinterleukin8(IL8).ItbindstoIL8withhighaffinity,andtransducesthesignalthroughaG-proteinactivatedsecondmessengersystem.Knockoutstudiesinmicesuggestedthatthisproteininhibitsembryonicoligodendrocyteprecursormigrationindevelopingspinalcord.Thisgene,IL8RB,ageneencodinganotherhighaffinityIL8receptor,aswellasIL8RBP,apseudogeneofIL8RB,formageneclusterinaregionmappedtochromosome2q33-q36.GeneSymbolIL8RACMKAR1CKR-1C-CC-C-CKR-1CXCR-1IL8RBAIL8R1CDw128aCD128CXCR1CD181GPCRClassAHostCellsChem-1ProteinTargetCXCR1/IL-8aTargetSub-FamilyChemokineUniProtNumberP25024UniProtSummaryFUNCTION:SwissProt:P25024#Receptortointerleukin-8,whichisapowerfulneutrophilschemotacticfactor.BindingofIL-8tothereceptorcausesactivationofneutrophils.ThisresponseismediatedviaaG-proteinthatactivateaphosphatidylinositol-calciumsecondmessengersystem.ThisreceptorbindstoIL-8withahighaffinityandtoMGSA(GRO)withalowaffinity.SIZE:350aminoacids;39791DaSUBCELLULARLOCATION:Cellmembrane;Multi-passmembraneprotein.SIMILARITY:SwissProt:P25024##BelongstotheG-proteincoupledreceptor1family.IncubationConditionsRECOMMENDEDASSAYCONDITIONS:Membranesaremixedwithradioactiveligandandunlabeledcompetitor(seeFigures1and2forconcentrationstested)inbindingbufferinanonbinding96-wellplate,andincubatedfor1-2h.Priortofiltration,anFC96-wellharvestplate(Milliporecat.#MAHFC1H)iscoatedwith0.33%polyethyleneiminefor30min,thenwashedwith50mMHEPES,pH7.4,0.5%BSA.Bindingreactionistransferredtothefilterplate,andwashed3times(1mLperwellperwash)withWashBuffer.Theplateisdriedandcounted.Bindingbuffer:50mMHepes,pH7.4,5mMMgCl2,1mMCaCl2,0.2%BSA,filteredandstoredat4°CRadioligand:[125I]Interleukin-8(PerkinElmer#NEX277)WashBuffer:50mMHepes,pH7.4,500mMNaCl,0.1%BSA,filteredandstoredat4°C.Onevialcontainsenoughmembranesforatleast200assays(units),whereanunitistheamountofmembranethatwillyieldgreaterthan5-foldsignal:backgroundwith125I-labeledIL-8at0.1nMPhysicochemicalInformationDimensionsMaterialsInformationMaterialsInformation