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- AntiHistone H3 Polyclonal Antibody (OAAI00717)
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- Rabbit AntiHA IgG: DyLight® 488, 100 μg
- Rabbit AntiHA IgG: DyLight® 488, 100 μg
联系方式
公司地址
苏州工业园区生物纳米园A4#216
联系电话
4000-520-616 / 18915418616
传真号码
0512-67156496
电子邮箱
info@ebiomall.com
公司网址
https://www.ebiomall.com
商品描述
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).
8.Ad Everest Biotech的山羊多克隆抗体以Everest Elite Grade或Aspiring Grade产品的形式发布，这取决于它们的测试性能和对靶蛋白的了解程度。Everest Biotech提供了圣克鲁斯多克隆产品系列的高质量替代品 精英级抗体保证按数据表上的规定工作，或退款。肽ELISA数据显示它们可以识别免疫肽。Western印迹数据显示生理相关组织或细胞系上的正确大小带。IHC / IF数据和特定出版物（如果有）。100％满意保证也可批量提供给化验开发人员 有抱负的抗体快速将市场上几乎没有或没有商业抗体可用且在文献中几乎没有表达数据的靶标引入市场。由肽ELISA数据支持。背景低但带大小错误或在可用于测试的常见细胞系和组织范围内没有信号。以大幅降低的价格出售，但我们不确定他们会在任何应用中识别天然蛋白质。100％满意保证奖励分享显示抗体成功发挥作用的数据-将您的数据提交到support@everestbiotech.com可获得您选择的免费抗体。免疫肽产生抗体的肽可用于封闭实验以100µg冻干肽的形式出售
