Everest Biotech/Human ANG ELISA Kit 1 Pate/2901690015/1 Pate,Everest Biotech,,Everest Biotech,everestbiotech,+,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：Everest Biotech/Human ANG ELISA Kit 1 Pate/2901690015/1 Pate

产品编号：2901690015

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

ForquantitativedetectionofhumanANGincellculturesupernates,serumandplasma(heparin,EDTA).

TypicalDataObtainedfromHumanANG

(TMBreactionincubateat37°Cfor15min)

Concentration(pg/ml)	0.0	78	156	313	625	1250	2500	5,000

O.D	0.007	0.255	0.500	0.757	1.194	1.660	1.787	1.966

TypicalHumanANGELISAKitStandardCurve

ThisstandardcurvewasgeneratedatEtonfordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.

Range78pg/ml-5000pg/ml

Sensitivity<12pg/ml

SpecificityNaturalandrecombinanthumanANG

Cross-reactivityNodetectablecross-reactivitywithotherrelevantproteins

Storage

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

Principle

Eton’shumanANGELISAKitwasbasedonstandardsandwichenzyme-linkedimmune-sorbentassaytechnology.AmonoclonalantibodyfrommousespecificforANGhasbeenprecoatedonto96-wellplates.Standards(E.coli,Q25-P147)andtestsamplesareaddedtothewells,abiotinylateddetectionpolyclonalantibodyfromgoatspecificforANGisaddedsubsequentlyandthenfollowedbywashingwithPBSorTBSbuffer.Avidin-Biotin-PeroxidaseComplexwasaddedandunboundconjugateswerewashedawaywithPBSorTBSbuffer.HRPsubstrateTMBwasusedtovisualizeHRPenzymaticreaction.TMBwascatalyzedbyHRPtoproduceabluecolorproductthatchangedintoyellowafteraddingacidicstopsolution.ThedensityofyellowisproportionaltothehumanANGamountofsamplecapturedinplate.

KitComponents

Description	Quantity

96-wellplateprecoatedwithanti-humanANGantibody	1

LyophilizedrecombinanthumanANGstandard	10ng/tube×2

Biotinylatedanti-humanANGantibody	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(50-500ng/ml).Theworkingdilutionis1:100.i.e.Add3μlsampleinto297μlsamplediluentbuffer.

Mediumtargetproteinconcentration(5-50ng/ml).Theworkingdilutionis1:10.i.e.Add25μlsampleinto225μlsamplediluentbuffer.

Lowtargetproteinconcentration(78-5000pg/ml).Theworkingdilutionis1:2.i.e.Add100μlsampleto100μlsamplediluentbuffer.

Verylowtargetproteinconcentration(≤78pg/ml).Nodilutionnecessary,ortheworkingdilutionis1:2.

3.ReagentPreparationandStorage

A.ReconstitutionofthehumanANGstandard:ANGstandardsolutionshouldbepreparednomorethan2hourspriortotheexperiment.TwotubesofANGstandard(10ngpertube)areincludedineachkit.Useonetubeforeachexperiment.

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

b.5000pg/ml→78pg/mlofhumanANGstandardsolutions:Label7Eppendorftubeswith5000pg/ml,2500pg/ml,1250pg/ml,625pg/ml,312pg/ml,156pg/ml,78pg/mlrespectively.Aliquot0.3mlofthesamplediluentbufferintoeachtube.Add0.3mloftheabove10,000pg/mlANGstandardsolutioninto1sttubeandmix.Transfer0.3mlfrom1sttubeto2ndtubeandmix.Transfer0.3mlfrom2ndtubeto3rdtubeandmix,andsoon.

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

B.Preparationofbiotinylatedanti-humanANGantibodyworkingsolution:Thesolutionshouldbepreparednomorethan2hourspriortotheexperiment.

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

b.Biotinylatedanti-humanANGantibodyshouldbedilutedin1:100withtheantibodydiluentbufferandmixedthoroughly.(i.e.Add1μlBiotinylatedanti-humanANGantibodyto99μlantibodydiluentbuffer.)

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.StandardANGdetectioncurveshouldbepreparedforeachexperiment.TheuserwilldecidesampledilutionfoldbycrudeestimationofANGamountinsamples.

1.Aliquot0.1mlperwellofthe4000pg/ml,2000pg/ml,1000pg/ml,500pg/ml,250pg/ml,125pg/ml,62.5pg/mlhumanANGstandardsolutionsintotheprecoated96-wellplate.Add0.1mlofthesamplediluentbufferintothecontrolwell(Zerowell).Add0.1mlofeachproperlydilutedsampleofhumancellculturesupernates,serumorplasma(heparin,EDTA)toeachemptywell.See“SampleDilutionGuideline”abovefordetails.ItisrecommendedthateachhumanANGstandardsolutionandeachsamplebemeasuredinduplicate.

2.Sealtheplatewiththecoverandincubateat37°Cfor90min.

3.Removethecover,discardplatecontent,andblottheplateontopapertowelsorotherabsorbentmaterial.DoNOTletthewellscompletelydryatanytime.

4.Add0.1mlofbiotinylatedanti-humanANGantibodyworkingsolutionintoeachwellandincubatetheplateat37°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.Add90μlofpreparedTMBcolordevelopingagentintoeachwellandincubateplateat37°Cindarkfor15-20min(Note:Forreferenceonly,theoptimalincubationtimeshouldbedeterminedbyenduser.AndtheshadesofbluecanbeseeninthewellswiththefourmostconcentratedhumanANGstandardsolutions;theotherwellsshownoobviouscolor).

9.Add0.1mlofpreparedTMBstopsolutionintoeachwell.Thecolorchangesintoyellowimmediately.

10.ReadtheO.D.absorbanceat450nminamicroplatereaderwithin30minafteraddingthestopsolution.

Forcalculation,(therelativeO.D.450)=(theO.D.450ofeachwell)–(theO.D.450ofZerowell).ThestandardcurvecanbeplottedastherelativeO.D.450ofeachstandardsolution(Y)vs.therespectiveconcentrationofthestandardsolution(X).ThehumanANGconcentrationofthesamplescanbeinterpolatedfromthestandardcurve.

Note:ifthesamplesmeasuredwerediluted,multiplythedilutionfactortotheconcentrationsfrominterpolationtoobtaintheconcentrationbeforedilution.

Summary

1.Addsamplesandstandardsandincubatetheplateat37°Cfor90min.Donotwash.

2.Addbiotinylatedantibodiesandincubatetheplateat37°Cfor60min.Washplate3timeswith0.01MTBS.

3.AddABCworkingsolutionandincubatetheplateat37°Cfor30min.Washplate5timeswith0.01MTBS.

4.AddTMBcolordevelopingagentandincubatetheplateat37°Cindarkfor15-20min.

5.AddTMBstopsolutionandread.

1.genetochromosomeband14q11,proximaltotheTcellreceptoralpha/deltalocus".AmJHumGenet47(6):973–81.

2."EntrezGene:ANGangiogenin,ribonuclease,RNaseAfamily,5".http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=283.

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。