品牌咨询
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
