Everest Biotech/Human BAFF ELISA Kit 1 Pate/2901980015/1 Pate,Everest Biotech,,Everest Biotech,everestbiotech,+,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：Everest Biotech/Human BAFF ELISA Kit 1 Pate/2901980015/1 Pate

产品编号：2901980015

市场价：¥4784.00

场地：美国(厂家直采)

产品分类：试剂盒>ELISA试剂盒>夹心法ELISA>

联系QQ：1570468124

电话号码：4000-520-616

邮箱： info@ebiomall.com

美元价：$460.00

品牌： Everest Biotech

公司：Everest Biotech

公司分类：

商品介绍

ForquantitativedetectionofhumanBAFFincellculturesupernates,serumandplasma(heparin,EDTA).

TypicalDataObtainedfromHumanBAFF

(TMBreactionincubateat37°Cfor20min)

Concentration(pg/ml)	0.0	62.5	125	250	500	1000	2000	4000

O.D	0.029	0.074	0.110	0.187	0.336	0.653	1.222	2.263

TypicalHumanBAFFELISAKitStandardCurve

ThisstandardcurvewasgeneratedatEtonfordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.

Range62.5pg/ml-4000pg/ml

Sensitivity<2pg/ml

SpecificityNaturalandrecombinanthumanBAFF

Cross-reactivityNodetectablecross-reactivitywithotherrelevantproteins

Storage

Storeat4°Cfor6months,at-20°Cfor12months.Avoidmultiplefreeze-thawcycles(Shippedwithwetice.)

Principle

Eton’shumanBAFFELISAKitwasbasedonstandardsandwichenzyme-linkedimmune-sorbentassaytechnology.AmonoclonalantibodyfrommousespecificforBAFFhasbeenprecoatedonto96-wellplates.Standardsandtestsamplesareaddedtothewells,abiotinylateddetectionpolyclonalantibodyfromgoatspecificforBAFFisaddedsubsequentlyandthenfollowedbywashingwithPBSorTBSbuffer.Avidin-Biotin-PeroxidaseComplexwasaddedandunboundconjugateswerewashedawaywithPBSorTBSbuffer.HRPsubstrateTMBwasusedtovisualizeHRPenzymaticreaction.TMBwascatalyzedbyHRPtoproduceabluecolorproductthatchangedintoyellowafteraddingacidicstopsolution.ThedensityofyellowisproportionaltothehumanBAFFamountofsamplecapturedinplate.

KitComponents

Description	Quantity

96-wellplateprecoatedwithanti-humanBAFFantibody	1

LyophilizedrecombinanthumanBAFFstandard	10ng/tube×2

Biotinylatedanti-humanBAFFantibody	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.Toavoidtousethereagentsfromdifferentbatchestogether.

8.Inordertoavoidmarginaleffectofplateincubationduetotemperaturedifference(reactionmaybestrongerinthemarginalwells),itissuggestedthatthedilutedABCandTMBsolutionwillbepre-warmedin37°Cfor30minbeforeusing.

Preparation

1.SamplePreparationandStorage

Storesamplestobeassayedwithin24hoursat2-8°C.Forlong-termstorage,aliquotandfreezesamplesat-20°C.Avoidrepeatedfreeze-thawcycles.

Cellculturesupernates:Removeparticulatesbycentrifugation,assayimmediatelyoraliquotandstoresamples

at-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.Add100μlsampleinto100μlsamplediluentbuffer.

Verylowtargetproteinconcentration(≤62.5pg/ml)Nodilutionnecessary,ortheworkingdilutionis1:2.

3.ReagentPreparationandStorage

A.ReconstitutionofthehumanBAFFstandard:BAFFstandardsolutionshouldbepreparednomorethan2hourspriortotheexperiment.TwotubesofBAFFstandard(10ngpertube)areincludedineachkit.Useonetubeforeachexperiment.

a.10,000pg/mlofhumanBAFFstandardsolution:Add1mlsamplediluentbufferintoonetube,keepthetubeatroomtemperaturefor10minandmixthoroughly.

b.4000pg/mlofhumanBAFFstandardsolution:Add0.4mloftheabove10ng/mlBAFFstandardsolutioninto0.6mlsamplediluentbufferandmixthoroughly.

c.2000pg/ml→62.5pg/mlofhumanBAFFstandardsolutions:Label6Eppendorftubeswith2000pg/ml,1000pg/ml,500pg/ml,250pg/ml,125pg/ml,62.5pg/mlrespectively.Aliquot0.3mlofthesamplediluentbufferintoeachtube.Add0.3mloftheabove4000pg/mlBAFFstandardsolutioninto1sttubeandmix.Transfer0.3mlfrom1sttubeto2ndtubeandmix.Transfer0.3mlfrom2ndtubeto3rdtubeandmix,andsoon.

Note:Thestandardsolutionsarebestusedwithin2hours.The10ng/mlstandardsolutionshouldbestoredat4°Cforupto12hours,orat-20°Cforupto48hours.Avoidrepeatedfreeze-thawcycles.

B.Preparationofbiotinylatedanti-humanBAFFantibodyworkingsolution:Thesolutionshouldbepreparednomorethan2hourspriortotheexperiment.

a.Thetotalvolumeshouldbe:0.1ml/wellx(thenumberofwells).(Allowing0.1-0.2mlmorethantotalvolume)

b.Biotinylatedanti-humanBAFFantibodyshouldbedilutedin1:100withtheantibodydiluentbufferandmixedthoroughly.(i.e.Add1μlBiotinylatedanti-humanBAFFantibodyto99μlantibodydiluentbuffer.)

C.PreparationofAvidin-Biotin-PeroxidaseComplex(ABC)workingsolution:Thesolutionshouldbepreparednomorethan1hourpriortotheexperiment.

a.Thetotalvolumeshouldbe:0.1ml/wellx(thenumberofwells).(Allowing0.1-0.2mlmorethantotalvolume)

b.Avidin-Biotin-PeroxidaseComplex(ABC)shouldbedilutedin1:100withtheABCdilutionbufferandmixedthoroughly.(i.e.Add1μlABCto99μlABCdiluentbuffer.)

AssayProcedure

TheABCworkingsolutionandTMBcolordevelopingagentmustbekeptwarmat37°Cfor30minbeforeuse.Whendilutingsamplesandreagents,theymustbemixedcompletelyandevenly.StandardBAFFdetectioncurveshouldbepreparedforeachexperiment.TheuserwilldecidesampledilutionfoldbycrudeestimationofBAFFamountinsamples.

1.Aliquot0.1mlperwellofthe4000pg/ml,2000pg/ml,1000pg/ml,500pg/ml,250pg/ml,125pg/ml,62.5pg/mlhumanBAFFstandardsolutionsintotheprecoated96-wellplate.Add0.1mlofthesamplediluentbufferintothecontrolwell(Zerowell).Add0.1mlofeachproperlydilutedsampleofhumancellculturesupernates,serumorplasma(heparin,EDTA)toeachemptywell.See“SampleDilutionGuideline”abovefordetails.ItisrecommendedthateachhumanBAFFstandardsolutionandeachsamplebemeasuredinduplicate.

2.Sealtheplatewiththecoverandincubateat37°Cfor90min.

3.Removethecover,discardplatecontent,andblottheplateontopapertowelsorotherabsorbentmaterial.DoNOTletthewellscompletelydryatanytime.

4.Add0.1mlofbiotinylatedanti-humanBAFFantibodyworkingsolutionintoeachwellandincubatetheplateat37°Cfor60min.

5.Washplate3timeswith0.01MTBSor0.01MPBS,andeachtimeletwashingbufferstayinthewellsfor1min.Discardthewashingbufferandblottheplateontopapertowelsorotherabsorbentmaterial.(PlateWashingMethod:Discardthesolutionintheplatewithouttouchingthesidewalls.Blottheplateontopapertowelsorotherabsorbentmaterial.Soakeachwellwithatleast0.3mlPBSorTBSbufferfor1~2minutes.RepeatthisprocesstwoadditionaltimesforatotalofTHREEwashes.Note:Forautomatedwashing,aspirateallwellsandwashTHREEtimeswithPBSorTBSbuffer,overfillingwellswithPBSorTBSbuffer.Blottheplateontopapertowelsorotherabsorbentmaterial.)

6.Add0.1mlofpreparedABCworkingsolutionintoeachwellandincubatetheplateat37°Cfor30min.

7.Washplate5timeswith0.01MTBSor0.01MPBS,andeachtimeletwashingbufferstayinthewellsfor1-2min.Discardthewashingbufferandblottheplateontopapertowelsorotherabsorbentmaterial.(SeeStep5forplatewashingmethod).

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品牌介绍

Everest Biotech公司成立于2000年，由英国牛津大学一批生物化学家创建。公司科学家团队具有丰富的抗体纯化及蛋白组学专业知识，雄厚的专业背景赋予了公司能为全球科研人员提供高质量、创新性抗体产品的能力。目前，Everest Biotech可提供3000余种抗体，涉及肿瘤、自身免疫、过敏、心血管疾病、传染病、代谢、神经科学等领域。所有抗体均由Everest Biotech自主研发生产、全部经过肽亲和纯化以保证最佳的特异性和纯度、经过免疫原肽的ELISA和WB实验验证; 由PhD科学家团队提供全方位技术支持服务。Everest Biotech为众多国际知名抗体公司提供OEM服务。