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.
