FORRESEARCHUSEONLY;NOTFORTHERAPEUTICORDIAGNOSTICAPPLICATI***!PLEASEREADTHROUGHENTIREPROCEDUREBEFOREBEGINNING!IntendeduseThisimmunoassaykitallowsfortheinvitroquantitativedeterminationofmouseinositol1,4,5,-trisphosphate,IP3concentrati***inserum,pla***a,tissuehomogenatesandotherbiologicalfluids.IntroductionInositoltrisphosphateorinositol1,4,5-trisphosphate(alsocommonlyknownastriphosphoinositol;abbreviatedInsP3orIP3),togetherwithdiacylglycerol,isasecondarymessengermoleculeusedinsignaltransductioninbiologicalcells.Itismadebyhydrolysisofphosphatidylinositol4,5-bisphosphate(PIP2),aphospholipidthatislocatedinthepla***amembrane,byphospholipaseC.IP3bindstoandactivatestheInsP3receptoronthemembraneoftheendopla***icreticulum(ER),andsarcopla***icreticulum(SR)opensacalciumchannel,resultinginthereleaseofCa2+intothecytopla***,andsarcopla***respectively.ThisincreaseinCa2+activatestheryanodinereceptor-operatedchannelontheSR,leadingtoafurtherincreaseintheCa2+.TestprincipleThemicrotiterplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictoIP3.Standa***orsamplesarethenaddedtotheappropriatemicrotiterplatewellswithabiotin-conjugatedpolyclonalantibodypreparationspecificforIP3and***idinconjugatedtoHorseradishPeroxidase(HRP)isaddedtoeachmicroplatewellandincubated.ThenaTMBsubstratesolutionisaddedtoeachwell.OnlythosewellsthatcontainIP3,biotin-conjugatedantibodyandenzyme-conjugated***idinwillexhibitachangeincolor.Theenzyme-substratereactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyataw***elengthof450nm&plu***n;2nm.TheconcentrationofIP3inthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.MaterialsandcomponentsReagentQuantityAssayplate1×20mlStandard2SampleDiluent1×20mlAssayDiluentA1×10mlAssayDiluentB1×10mlDetectionReagentA1×120μlDetectionReagentB1×120μlWashBuffer(25xconcentrate)1×30mlSubstrate1×10mlStopSolution1×10mlPlatesealerfor96wells5Instruction1OthersuppliesrequiredLuminometer.Pipettesandpipettetips.EPtubeDeionizedordistilledwater.SamplecollectionandstorageSerum-Useaserumseparatortube(SST)andallowsamplestoclotfor30minutesbeforecentrifugationfor15minutesatapproximately1000×g.Removeserumandassayimmediatelyoraliquotandstoresamplesat-20℃or-80℃.Pla***a-Collectpla***ausingEDTAorheparinasananticoagulant.Centrifugesamplesfor15minutesat1000×gat2-8℃within30minutesofcollection.Storesamplesat-20℃or-80℃.***oidrepeatedfreeze-thawcycles.Tissuehomogenates-Thepreparationoftissuehomogenateswillvarydependingupontissuetype.Forthisassay,tissuewasrinsedwith1XPBStoremoveexcessblood,homogenizedin20mLof1XPBSandstoredovernightat≤-20°C.Aftertwofreeze-thawcycleswereperformedtobreakthecellmembranes,thehomogenateswerecentrifugedfor5minutesat5000xg.Removethesupernateandassayimmediatelyoraliquotandstoreat≤-20°C.Otherbiologicalfluids-Removeparticulatesbycentrifugationandassayimmediatelyoraliquotandstoresamplesat-20℃or-80℃.***oidrepeatedfreeze-thawcycles.Note:Serum,pla***aandtissuehomogenatestobeusedwithin7daysmaybestoredat2-8℃,otherwisesamplesmuststoredat-20℃(≤1months)or-80℃(≤2months)to***oidlossofbioactivityandcontamination.***oidfreeze-thawcycles.Whenperformingtheassayslowlybringsamplestoroomtemperature.DONOTUSEHEAT-TREATEDSPECIMENS.Limitati***oftheprocedure1.Thekitshouldnotbeusedbeyondtheexpirationdateonthekitlabel.2.Donotmixorsubstitutereagentswiththosefromotherlotsorsources.3.Ifsamplesgeneratevalueshigherthanthehigheststandard,furtherdilutethesampleswiththeAssayDiluentandrepeattheassay.Anyvariationinstandarddiluent,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecancausevariationinbinding.4.Thisassayisdesignedtoeliminateinterferencebysolublereceptors,ligands,bindingproteins,andotherfactorspresentinbiologicalsamples.Untilallfactorsh***ebeentestedintheQuantikineImmunoassay,thepossibilityofinterferencecannotbeexcluded.说明书Email:info@索取;13801829628定货；HR-M0227Mouseinositol1,4,5,-trisphosphate,IP3ELISAKit)