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蚂蚁淘在线 / 品牌中心 / Everest Biotech / Everest Biotech/Human Azurocidin ELISA Kit 1 plate/2904450015/1 plate
商品描述
ForquantitativedetectionofhumanAzurocidinincellculturesupernates,serumandplasma(heparin,EDTA).TypicalDataObtainedfromHumanAzurocidin
(TMBreactionincubateat37°Cfor25min)
Concentration(pg/ml) | 0.0 | 62.5 | 125 | 250 | 500 | 1000 | 2000 | 4000 |
O.D | 0.015 | 0.046 | 0.070 | 0.121 | 0.293 | 0.650 | 1.514 | 2.260 |
TypicalHumanAzurocidinELISAKitStandardCurve
ThisstandardcurvewasgeneratedatEtonfordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.Range62.5pg/ml-4000pg/ml
Sensitivity<10pg/ml
SpecificityNaturalandrecombinanthumanAzurocidin
Cross-reactivityNodetectablecross-reactivitywithotherrelevantproteins
Storage
Storeat4°Cfor6months,at-20°Cfor12months.Avoidmultiplefreeze-thawcycles(Shippedwithwetice.)
Precision
Intra-AssayPrecision(Precisionwithinanassay)Threesamplesofknownconcentrationweretestedononeplatetoassessintra-assayprecision.
Inter-AssayPrecision(Precisionbetweenassays)Threesamplesofknownconcentrationweretestedinseparateassaystoassessinter-assayprecision.
Intra-AssayPrecision | Inter-AssayPrecision | |||||
Sample | 1 | 2 | 3 | 1 | 2 | 3 |
n | 16 | 16 | 16 | 24 | 24 | 24 |
Mean(pg/ml) | 675 | 1847 | 2816 | 873 | 2020 | 3174 |
StandardDeviation | 28.35 | 94.2 | 149.2 | 48.9 | 129.3 | 238.1 |
CV(%) | 4.2 | 5.1 | 5.3 | 5.6 | 6.4 | 7.5 |
Principle
Eton’shumanAzurocidinELISAKitwasbasedonstandardsandwichenzyme-linkedimmune-sorbentassaytechnology.AmonoclonalantibodyfrommousespecificforAzurocidinhasbeenprecoatedonto96-wellplates.Standards(NSO,I27-P248)andtestsamplesareaddedtothewells,abiotinylateddetectionpolyclonalantibodyfromgoatspecificforAzurocidinisaddedsubsequentlyandthenfollowedbywashingwithPBSorTBSbuffer.Avidin-Biotin-PeroxidaseComplexwasaddedandunboundconjugateswerewashedawaywithPBSorTBSbuffer.HRPsubstrateTMBwasusedtovisualizeHRPenzymaticreaction.TMBwascatalyzedbyHRPtoproduceabluecolorproductthatchangedintoyellowafteraddingacidicstopsolution.ThedensityofyellowisproportionaltothehumanAzurocidinamountofsamplecapturedinplate.
KitComponents
Description | Quantity |
96-wellplateprecoatedwithanti-humanAzurocidinantibody | 1 |
LyophilizedrecombinanthumanAzurocidinstandard | 10ng/tube×2 |
Biotinylatedanti-humanAzurocidinantibody | 130μl(dilution1:100) |
Avidin-Biotin-PeroxidaseComplex(ABC) | 130μl(dilution1:100) |
Samplediluentbuffer | 30ml |
Antibodydiluentbuffer | 12ml |
ABCdiluentbuffer | 12ml |
TMBcolordevelopingagent | 10ml |
TMBstopsolution | 10ml |
MaterialRequiredButNotProvided
1.Microplatereaderinstandardsize.
2.Automatedplatewasher.
3.AdjustablePipettesandpipettetips.Multichannelpipettesarerecommendedintheconditionoflargeamountofsamplesinthedetection.
4.CleantubesandEppendorftubes.
5.Washingbuffer(neutralPBSorTBS).
Preparationof0.01MTBS:Add1.2gTris,8.5gNacl;450μlofpurifiedaceticacidor700μlofconcentratedhydrochloricacidto1000mlH2OandadjustpHto7.2-7.6.Finally,adjustthetotalvolumeto1L.
Preparationof0.01MPBS:Add8.5gsodiumchloride,1.4gNa2HPO4and0.2gNaH2PO4to1000mldistilledwaterandadjustpHto7.2-7.6.Finally,adjustthetotalvolumeto1L.
NoticeforApplicationofKit
1.Toinspectthevalidityofexperimentoperationandtheappropriatenessofsampledilutionproportion,pilotexperimentusingstandardsandasmallnumberofsamplesisrecommended.
2.TheTMBColorDevelopingagentiscolorlessandtransparentbeforeusing,contactusfreelyifitisnotthecase.
3.BeforeusingtheKit,spintubesandbringdownallcomponentstothebottomoftubes.
4.Duplicatewellassayisrecommendedforbothstandardandsampletesting.
5.Don’tlet96-wellplatedry,fordryplatewillinactivateactivecomponentsonplate.
6.Don’treusetipsandtubestoavoidcrosscontamination.
7.Avoidusingthereagentsfromdifferentbatchestogether.
8.Inordertoavoidmarginaleffectofplateincubationduetotemperaturedifference(reactionmaybestrongerinthemarginalwells),itissuggestedthatthedilutedABCandTMBsolutionwillbepre-warmedin37℃for30minbeforeusing.
Preparation
1.SamplePreparationandStorage
Storesamplestobeassayedwithin24hoursat2-8°C.Forlong-termstorage,aliquotandfreezesamplesat-20°C.Avoidrepeatedfreeze-thawcycles.
Cellculturesupernates:Removeparticulatesbycentrifugation,assayimmediatelyoraliquotandstoresamplesat
-20°C.
Serum:Allowtheserumtoclotinaserumseparatortube(about4hours)atroomtemperature.Centrifugeatapproximately1000Xgfor15min.Analyzetheserumimmediatelyoraliquotandstoresamplesat-20°C.
Plasma:CollectplasmausingheparinorEDTAasananticoagulant.Centrifugefor15minat1500xgwithin30minofcollection.Assayimmediatelyoraliquotandstoresamplesat-20°C.
2.SampleDilutionGuideline
Theuserneedstoestimatetheconcentrationofthetargetproteininthesampleandselectaproperdilutionfactorsothatthedilutedtargetproteinconcentrationfallsnearthemiddleofthelinearregimeinthestandardcurve.Dilutethesampleusingtheprovideddiluentbuffer.Thefollowingisaguidelineforsampledilution.Severaltrialsmaybenecessaryinpractice.Thesamplemustbewellmixedwiththediluentsbuffer.
Hightargetproteinconcentration(40-400ng/ml).Theworkingdilutionis1:100.i.e.Add3μlsampleinto297μlsamplediluentbuffer.
Mediumtargetproteinconcentration(4-40ng/ml).Theworkingdilutionis1:10.i.e.Add25μlsampleinto225μlsamplediluentbuffer.
Lowtargetproteinconcentration(62.5-4000pg/ml).Theworkingdilutionis1:2.i.e.Add100μlsampleto100μlsamplediluentbuffer.
VeryLowtargetproteinconcentration(≤62.5pg/ml).Nodilutionnecessary,ortheworkingdilutionis1:2.
3.ReagentPreparationandStorage
A.Reconstitutionofthehu Everest Biotech自2000年以来，我们一直是抗肽和抗原亲和纯化山羊多克隆抗体的专家。我们的抗体具有100％的满意保证-它们可在您的实验室或您的退款中发挥作用。 LifeSpan BioSciences，Inc.收购了Everest Biotech。 在此处查找使用Everest抗体的最新出版物盖玻片 用于悬浮细胞免疫荧光
