| ApplicationNotes | - Limitedbythecurrentconditionandscientifictechnology,wecannotcompletelyconductthecomprehensiveidentificationandanalysisontherawmaterialprovidedbysuppliers.Sotheremightbesomequalitativeandtechnicalriskstousethekit.
- Thefinalexperimentalresultswillbecloselyrelatedtovalidityoftheproducts,operationskillsoftheendusersandtheexperimentalenvironments.Pleasemakesurethatsufficientsamplesareavailable.
- Kitsfromdifferentbatchesmaybealittledifferentindetectionrange,sensitivityandcolordevelopingtime.
- Donotmixorsubstitutereagentsfromonekitlottoanother.Useonlythereagentssuppliedbymanufacturer.
- Protectallreagentsfromstronglightduringstorageandincubation.Allthebottlecapsofreagentsshouldbecoveredtightlytopreventtheevaporationandcontaminationofmicroorganism.
- Theremaybesomefoggysubstanceinthewellswhentheplateisopenedatthefirsttime.Itwillnothaveanyeffectonthefinalassayresults.Donotremovemicrotiterplatefromthestoragebaguntilneeded.
- Wrongoperationsduringthereagentspreparationandloading,aswellasincorrectparametersettingfortheplatereadermayleadtoincorrectresults.Amicroplateplatereaderwithabandwidthof10nmorlessandanopticaldensityrangeof0-3O.D.orgreaterat450±10nmwavelengthisacceptableforuseinabsorbancemeasurement.Pleasereadtheinstructioncarefullyandadjusttheinstrumentpriortotheexperiment.
- Eventhesameoperatormightgetdifferentresultsintwoseparateexperiments.Inordertogetbetterreproducibleresults,theoperationofeverystepintheassayshouldbecontrolled.FurThermore,apreliminaryexperimentbeforeassayforeachbatchisrecommended.
- EachkithasbeenstrictlypassedQ.Ctest.However,resultsfromendusersmightbeinconsistentwithourin-housedataduetosomeunexpectedtransportationconditionsordifferentlabequipments.Intra-assayvarianceamongkitsfromdifferentbatchesmightarisefromabovefactors,too.
- Kitsfromdifferentmanufacturersforthesameitemmightproducedifferentresults,sincewehavenotcomparedourproductswithothermanufacturers.
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| Comment | Informationonstandardmaterial:
Thestandardmightberecombinantproteinornaturalprotein,thatwilldependonthespecifickit.Moreover,theexpressionsystemisE.colioryeastormammalcell.Thereis0.05%proclin300inthestandardaspreservative.
Informationonreagents:
Thestopsolutionusedinthekitissulfuricacidwithconcentrationof1mol/L.AndthewashsolutionisTBS.Thestandarddiluentcontains0.02%sodiumazide,assaydiluentAandassaydiluentBcontain0.01%sodiumazide.SomekitscancontainisBSAinthem.
Informationonantibodies:
Theprovidedantibodiesandtheirhostvaryindifferentkits. |
| SampleVolume | 100μL |
| AssayTime | 3h |
| Plate | Pre-coated |
| Protocol | Themicroplateprovidedinthiskithasbeenpre-coatedwithanantibodyspecifictoHypoxiaInducibleFactor1Alpha(HIF1a).Standardsorsamplesarethenaddedtotheappropriatemicroplatewellswithabiotin-conjugatedantibodyspecifictoHypoxiaInducibleFactor1Alpha(HIF1a).Next,AvidinconjugatedtoHorseradishPeroxidase(HRP)isaddedtoeachmicroplatewellandincubated.ThenthemixtureofsubstrateAandBisaddedtogenerateglowlightemissionkinetics.Uponplatedevelopment,theintensityoftheemittedlightisproportionaltotheHypoxiaInducibleFactor1Alpha(HIF1a)levelinthesampleorstandard., |
| ReagentPreparation | - Bringallkitcomponentsandsamplestoroomtemperature(18-25°C)beforeuse.
- Standard-ReconstitutetheStandardwith1.0mLofStandardDiluent,keptfor10minutesatroomtemperature,shakegently(nottofoam).Theconcentrationofthestandardinthestocksolutionis10ng/mL.Pleaseprepare7tubescontaining0.5mLStandardDiluentandproduceadoubledilutionseries.Mixeachtubethoroughlybeforethenexttransfer.Setup7pointsofdilutedstandardsuchas10ng/mL,5ng/mL,2.5ng/mL,1.25ng/mL,0.625ng/mL,0.312ng/mL,0.156ng/mL,andthelastEPtubeswithStandardDiluentistheblankas0ng/mL.
- AssayDiluentAandAssayDiluentB-Dilute6mLofAssayDiluentAorBConcentrate(2×)with6mLofdeionizedordistilledwatertoprepare12mLofAssayDiluentAorB.Thepreparedworkingdilutioncannotbefrozen.(Infact,morethan6mLAssayDiluentAandAssayDiluentBarecontainedinthebottles.Therefore,ineverytest,pleasepreciselypipetterequiredamountofDiluentandmakedoubledilutioninanewcontainer.Thepreparedworkingdilutioncannotbefrozen.)
- DetectionReagentAandDetectionReagentB-BrieflyspinorcentrifugethestockDetectionAandDetectionBbeforeuse.DilutetotheworkingconcentrationwithworkingAssayDiluentAorB,respectively(1:100).
- WashSolution-Dilute20mLofWashSolutionconcentrate(30×)with580mLofdeionizedordistilledwatertoprepare600mLofWashSolution(1×).
- SubstrateworkingSolution-MixthesubstrateAandBbytheratioof99:1tomakethesubstrateworkingsolution.Mixthoroughly.Forexample,prepare1,000µLSubstrateworkingSolutionwith990µLSubstrateA+10µLSubstrateB.
Note:- Makingserialdilutioninthewellsdirectlyisnotpermitted.
- Preparestandardwithin15minutesbeforeassay.Pleasedonotdissolvethereagentsat37°Cdirectly.
- PleasecarefullyreconstituteStandardsorworkingDetectionReagentAandBaccordingtotheinstruction,andavoidfoamingandmixgentlyuntilthecrystalsarecompletelydissolved.Tominimizeimprecisioncausedbypipetting,usesmallvolumesandensurethatpipettorsarecalibrated.Itisrecommendedtosuckmorethan10µLforoncepipetting.
- ThereconstitutedStandards,DetectionReagentAandDetectionReagentBcanbeusedonlyonce.
- PrepareSubstrateworkingSolutionwithin15minutesbeforeassay.
- IfcrystalshaveformedintheWashSolutionconcentrate(30×),warmtoroomtemperatureandmixgentlyuntilthecrystalsarecompletelydissolved.
- Contaminatedwaterorcontainerforreagentpreparationwillinfluencethedetectionresult.
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| SampleCollection | Serum:Allowsamplestoclotfortwohoursatroomtemperatureorovernightat4°Cbeforecentrifugationfor20minutesatapproximately1000×g.Assayimmediatelyorstoresamplesinaliquotat-20°Cor-80°C.Avoidrepeatedfreeze/thawcycles.
Plasma:CollectplasmausingEDTAorheparinasananticoagulant.Centrifugesamplesfor15minutesat1000×gwithin30minutesofcollection.Removeplasmaandassayimmediatelyorstoresamplesinaliquotat-20°Cor-80°C.Avoidrepeatedfreeze/thawcycles.
BiologicalFluids:Centrifugesamplesfor20minutesat1000×g.Removeparticulatesandassayimmediatelyorstoresamplesinaliquotat-20°Cor-80°Cforlateruse.Avoidrepeatedfreeze/thawcycles. |
| SamplePreparation | - Weareonlyresponsibleforthekititself,butnotforthesamplesconsumedduringtheassay.Theusershouldcalculatethepossibleamountofthesamplesusedinthewholetest.Pleasereservesufficientsamplesinadvance.
- Pleasepredicttheconcentrationbeforeassaying.Ifvaluesforthesearenotwithintherangeofthestandardcurve,usersmustdeterminetheoptimalsampledilutionsfortheirparticularexperiments.Sampleshouldbedilutedby0.01mol/LPBS(PH=7.0-7.2).
- Ifthesamplesarenotindicatedinthemanual,apreliminaryexperimenttodeterminethevalidityofthekitisnecessary.
- TissueorcellextractionsamplespreparedbychemicallysisbuffermaycauseunexpectedELISAresultsduetotheimpactsfromcertainchemicals.
- Duetothepossibilityofmismatchingbetweenantigenfromotheroriginandantibodyusedinourkits(e.g.antibodytargetsconformationalepitoperatherthanlinearepitope),somenativeorrecombinantproteinsfromothermanufacturersmaynotberecognizedbyourproducts.
- InfluencedbythefactorsincludingcellviABIlity,cellnumberorsamplingtime,samplesfromcellculturesupernatantmaynotbedetectedbythekit.
- Freshsampleswithoutlongtimestorageisrecommendedforthetest.Otherwise,proteindegradationanddenaturalizationmayoccurinthosesamplesandfinallyleadtowrongresults.
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| AssayProcedure | - Prepareallreagents,samplesandstandards,
- Add100μLstandardorsampletoeachwell.Incubate2hoursat37 °C,
- Aspirateandadd100μLpreparedDetectionReagentA.Incubate1hourat37 °C,
- Aspirateandwash3times,
- Add100μLpreparedDetectionReagentB.Incubate30 minutesat37 °C,
- Aspirateandwash5times,
- Add100μLSubstrateSolution.Incubate10 minutesat37 °C,
- ReadRLUvalueimmediately.
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| CalculationofResults | Averagetheduplicatereadingsforeachstandard,control,andsamplesandsubtracttheaveragezerostandardrelativelightunit(RLU).Createastandardcurveonlog-loggraphpaper,withHIF1aconcentrationonthey-axisandtheRLUvalueonthex-axis.Drawthebestfitstraightlinethroughthestandardpointsanditcanbedeterminedbyregressionanalysis.Usingsomeplotsoftware,suchascurveexpert1.30,isalsorecommended.Ifsampleshavebeendiluted,theconcentrationreadfromthestandardcurvemustbemultipliedbythedilutionfactor.
Inordertomakethecalculationeasier,weplottheRLUvalueofthestandard(X-axis)againsttheknownconcentrationofthestandard(Y-axis),althoughconcentrationisindeedtheindependentvariablewhileRLUvalueisthedependentvariable.Further,inthispart,inordertohelpthecustomerperformtheassaymorevisual,weprovidethecustomerwiththerawdata(notthelogofdata).However,plottinglogofthedatatoconstructthecurvewillberecommended.TheRLUvaluesofthestandardcurvemayvaryaccordingtotheconditionsofassayperformance(e.g.operator,pipettingtechnique,washingtechniqueortemperatureeffects).Thiscurveisprovidedfordemonstrationonly.Thecustomersshouldestablishtheirownstandardcurveforeachtestconducted. |
| AssayPrecision | Intra-assayPrecision(Precisionwithinanassay):3sampleswithlow,middleandhighlevelHypoxiaInducibleFactor1Alpha(HIF1a)weretested20timesononeplate,respectively.
Inter-assayPrecision(Precisionbetweenassays):3sampleswithlow,middleandhighlevelHypoxiaInducibleFactor1Alpha(HIF1a)weretestedon3differentplates,8replicatesineachplate.
CV(%)=SD/meanX100
Intra-Assay:CV<10%
Inter-Assay:CV<12%
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| Restrictions | ForResearchUseonly |
