Everest Biotech/Human ADAM12 ELISA Kit 1 Plate/2903500015/1 Plate,Everest Biotech,,Everest Biotech,everestbiotech,+,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：Everest Biotech/Human ADAM12 ELISA Kit 1 Plate/2903500015/1 Plate

产品编号：2903500015

市场价：¥4461.60

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：$429.00

品牌： Everest Biotech

公司：Everest Biotech

公司分类：

商品介绍

TypicalDataObtainedfromHumanADAM12

(TMBreactionincubateat37°Cfor20min)

Concentration(pg/ml)	0	156	312	625	1250	2500	5000	10,000

O.D	0.006	0.060	0.113	0.209	0.437	0.853	1.599	2.237

TypicalHumanADAM12ELISAKitStandardCurve

ThisstandardcurvewasgeneratedatEtonfordemonstrationpurposeonly.Astandardcurvemustberunwitheachassay.

Range156pg/ml-10,000pg/ml

Sensitivity<10pg/ml

SpecificityNaturalandrecombinanthumanADAM12

Cross-reactivityNodetectablecross-reactivitywithotherrelevantproteins

Storage

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

Principle

Eton’shumanADAM12ELISAKitwasbasedonstandardsandwichenzyme-linkedimmune-sorbentassaytechnology.AmonoclonalantibodyfrommousespecificforADAM12hasbeenprecoatedonto96-wellplates.Standards(CHO,R29-S513)andtestsamplesareaddedtothewells,abiotinylateddetectionpolyclonalantibodyfromgoatspecificforADAM12isaddedsubsequentlyandthenfollowedbywashingwithPBSorTBSbuffer.Avidin-Biotin-PeroxidaseComplexwasaddedandunboundconjugateswerewashedawaywithPBSorTBSbuffer.HRPsubstrateTMBwasusedtovisualizeHRPenzymaticreaction.TMBwascatalyzedbyHRPtoproduceabluecolorproductthatchangedintoyellowafteraddingacidicstopsolution.ThedensityofyellowisproportionaltothehumanADAM12amountofsamplecapturedinplate.Thiskitrecognizespro-ADAM12,matureADAM12,andADAM12/TIMP-3complex.

KitComponents

Description	Quantity

96-wellplateprecoatedwithanti-humanADAM12antibody	1

LyophilizedrecombinanthumanADAM12standard	10ng/tube×2

Biotinylatedanti-humanADAM12antibody	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:Collectplasmausingheparinasananticoagulant.Centrifugefor15minat1500xgwithin30minofcollection.Assayimmediatelyoraliquotandstoresamplesat-20°C.

Urine:Asepticallycollectthefirsturineoftheday,micturatedirectlyintoasterilecontainer.Removeparticularimpuritiesbycentrifugation,assayimmediatelyoraliquotandstoresamplesat-20°C.

2.SampleDilutionGuideline

Theuserneedstoestimatetheconcentrationofthetargetproteininthesampleandselectaproperdilutionfactorsothatthedilutedtargetproteinconcentrationfallsnearthemiddleofthelinearregimeinthestandardcurve.Dilutethesampleusingtheprovideddiluentbuffer.Thefollowingisaguidelineforsampledilution.Severaltrialsmaybenecessaryinpractice.Thesamplemustbewellmixedwiththediluentsbuffer.

Hightargetproteinconcentration(100-1000ng/ml).Theworkingdilutionis1:100.i.e.Add1μlsampleinto99μlsamplediluentbuffer.

Mediumtargetproteinconcentration(10-100ng/ml).Theworkingdilutionis1:10.i.e.Add10μlsampleinto90μlsamplediluentbuffer.

Lowtargetproteinconcentration(156-10,000pg/ml).Theworkingdilutionis1:2.i.e.Add50μlsampleto50μlsamplediluentbuffer.

Verylowtargetproteinconcentration(≤156pg/ml).Nodilutionnecessary,ortheworkingdilutionis1:2.

3.ReagentPreparationandStorage

A.ReconstitutionofthehumanADAM12standard:ADAM12standardsolutionshouldbepreparednomorethan2hourspriortotheexperiment.TwotubesofADAM12standard(10ngpertube)areincludedineachkit.Useonetubeforeachexperiment.

a.10,000pg/mlofhumanADAM12standardsolution:Add1mlsamplediluentbufferintoonetube,keepthetubeatroomtemperaturefor10minandmixthoroughly.

b.5000pg/ml156pg/mlofhumanADAM12standardsolutions:Label6Eppendorftubeswith5000pg/ml,2500pg/ml,1250pg/ml,625pg/ml,312pg/ml,156pg/mlrespectively.Aliquot0.3mlofthesamplediluentbufferintoeachtube.Add0.3mloftheabove10,000pg/mlADAM12standardsolutioninto1sttubeandmix.Transfer0.3mlfrom1sttubeto2ndtubeandmix.Transfer0.3mlfrom2ndtubeto3rdtubeandmix,andsoon.

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

B.Preparationofbiotinylatedanti-humanADAM12antibodyworkingsolution:Thesolutionshouldbepreparednomorethan2hourspriortotheexperiment.

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

b.Biotinylatedanti-humanADAM12antibodyshouldbedilutedin1:100withtheantibodydiluentbufferandmixedthoroughly.(i.e.Add1μlBiotinylatedanti-humanADAM12antibodyto99μlantibodydiluentbuffer.)

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.StandardADAM12detectioncurveshouldbepreparedforeachexperiment.TheuserwilldecidesampledilutionfoldbycrudeestimationofADAM12amountinsamples.

1.Aliquot0.1mlperwellofthe10,000pg/ml,5000pg/ml,2500pg/ml,1250pg/ml,625pg/ml,312pg/ml,156pg/mlhumanADAM12standardsolutionsintotheprecoated96-wellplate.Add0.1mlofthesamplediluentbufferintothecontrolwell(Zerowell).Add0.1mlofeachproperlydilutedsampleofhumancellculturesupernates,serum,plasma(heparin)orurinetoeachemptywell.See“SampleDilutionGuideline”abovefordetails.ItisrecommendedthateachhumanADAM12standardsolutionandeachsamplebemeasuredinduplicate.

2.Sealtheplatewiththecoverandincubateat37°Cfor90min.

3.Removethecover,discardplatecontent,andblottheplateontopapertowelsorotherabsorbentmaterial.DoNOTletthewellscompletelydryatanytime.

4.Add0.1mlofbiotinylatedanti-humanADAM12antibodyworkingsolutionintoeachwellandincubatetheplateat37°Cfor60min.

5.Washplate3timeswith0.01MTBSor0.01MPBS,andeachtimeletwashingbufferstayinthewellsfor

min.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°Cindarkfor20-25min(Note:Forreferenceonly,theoptimalincubationtimeshouldbedeterminedbyenduser.AndtheshadesofbluecanbeseeninthewellswiththefourmostconcentratedhumanADAM12standardsolutions;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).ThehumanADAM12concentrationofthesamplescanbeinterpolatedfromthestandardcurve.

Note:ifthesamplesmeasuredwerediluted,multiplythedilutionfactortotheconcentrationsfrominterpolationtoobtaintheconcentrationbeforedilution.

Summary

1.Addsamplesandstandardsandincubatetheplateat37°Cfor90min.Donotwash.

2.Addbiotinylatedantibodiesandincubatetheplateat37°Cfor60min.Washplate3timeswith0.01MTBS.

3.AddABCworkingsolutionandincubatetheplateat37°Cfor30min.Washplate5timeswith0.01MTBS.

4.AddTMBcolordevelopingagentandincubatetheplateat37°Cindarkfor20-25min.

5.AddTMBstopsolutionandread.

1.Galliano,M.-F.,Huet,C.,Frygelius,J.,Polgren,A.,Wewer,U.M.,Engvall,E.BindingofADAM12,aMarkerofskeletalmuscleregeneration,tothemuscle-specificactin-bindingprotein,alpha-actinin-2,isrequiredformyoblastfusion.J.Biol.Chem.275:13933-13939,2000.

2.Gilpin,B.J.,Loechel,F.,Mattei,M.-G.,Engvall,E.,Albrechtsen,R.,Wewer,U.M.Anovel,secretedformofhumanADAM12(meltrinalpha)provokesmyogenesisinvivo.J.Biol.Chem.273:157-166,1998.

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。