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Contents:MouseTNFReadySETGo!ELISAKitWithPreCoatedPlates Catalog Number: 88-7342 Sensitivity: 8pg/ml Standard Curve Range: 81000pg/ml Sizes:2precoatedplates,10precoatedplates

MouseTNFReadySETGo!ELISAKitStandardCurve

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Cat. No. 887342 Format MouseTNFa(TumorNecrosisFactoralpha,TNFalpha,TNFa)ELISAReadySET Go!Kit(withPreCoatedPlates) Excite Emit Reported (nm) (nm) Applications N/A N/A ELISAkit

Questions?Pleaseconsultouranswerstofrequentlyaskedquestionsathttp://www.ebioscience.com/faq. Description TheMouseTNFReadySETGo!ELISAKit(includingprecoatedELISAplates)containsthenecessaryreagents,buffersanddiluentsfor performingquantitativeenzymelinkedimmunosorbentassays(ELISA).ThisELISAkitisspecificallyengineeredforaccurateandprecise measurementofmouseTNFproteinlevelsfromsamplesincludingserum,plasma,andsupernatantsfromcellcultures.Recognitionof mouseTNFbythisassayisnotinterferedwithby1000xexcesssolubleTNFreceptortypeIorII. Components 1. 2. 3. 4. 5. 6. 7. 8. 9. PrecoatedELISAPlate,cloneTN3.19 DetectionAntibody:Pretitrated,biotinconjugatedantibody,clonePolyclonal Standard:Recombinantcytokineforgeneratingstandardcurveandcalibratingsamples AssayDiluent:5Xconcentrated ELISAWashBufferPowder Detectionenzyme:pretitratedAvidinHRP SubstrateSolution:Tetramethylbenzidine(TMB)SubstrateSolution StopSolution:5mlsof1Xsolutionperplate CertificateofAnalysis:Lotspecificinstructionsfordilutionofantibodiesandstandards

Applications Reported For research use only, not for diagnostic or therapeutic use.

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Other Materials Needed Pipettesandpipettors Refrigerator 96wellELISAplatereader(microplatespectrophotometer) ELISAplatewasher Stability ThisELISAkitisguaranteedtoperformasspecifiedatleast12monthsfromdateofreceiptifstoredandhandledasinstructed accordingtothisdatasheetandtheCertificateofAnalysis,whichisincludedwiththereagents. Storage Instructions for Cytokine Standards Thefrozencytokinestandardisalreadyaliquotedat20lpervial.Uponreceipt,frozencytokinestandardshouldbeimmediatelystored at80Cstableforatleast12months.Afterthawing,quickspinvialpriortoopening.Donotrealiquotintosmallerfractions.These aresingleusevials.Useonetimeanddiscard.Fordilutionofthestandard,pleaseseeinstructionsontheCertificateofAnalysisand followtheseaswritten. Storage Instructions for other kit reagents Storeat4C. IMPORTANT NOTE: Be certain that no sodium azide is present in the solutions used in this assay, as this inhibits HRP enzyme activity. Time Requirements 4hourincubations 1hourwashingandanalyzingsamples

Experimental Procedure

1.

2. 3. 4. 5.

UsingAssayDiluent,dilutestandardsasnotedontheCertificateofAnalysis(CofA).Add100l/wellofstandardtothe appropriatewells.Perform2foldserialdilutionsofthetopstandardstomakethestandardcurve.Add100l/wellofyour samplestotheappropriatewells.Coverorsealtheplateandincubateatroomtemperaturefor2hours(orovernightat4Cfor maximalsensitivity). Aspiratewellsandwash5timeswith>250ul/wellWashBuffer(dilutedto1X).Allowingtimeforsoaking(~1minute)during eachwashstepincreasestheeffectivenessofthewashes.Blotplateonabsorbentpapertoremoveanyresidualbuffer. Add100l/wellofdetectionantibodydilutedin1XAssayDiluent(diluteasnotedonCofA).Sealtheplateandincubateatroom temperaturefor1hour. Aspirate/washasinstep2.Repeatforatotalof5washes. Add100l/wellofAvidinHRP*dilutedin1XAssayDiluent(diluteasnotedonCofA).Sealtheplateandincubateatroom temperaturefor30minutes. *Important:Donotincludesodiumazideinanybuffers,asthiswillinactivatetheHRP. Aspirateandwashasinstep2.Inthiswashstep,soakwellsinWashBufferfor1to2minutespriortoaspiration.Repeatfora totalof7washes. Add100l/wellofSubstrateSolutiontoeachwell.Incubateplateatroomtemperaturefor15minutes. Add50lofStopSolutiontoeachwell. Readplateat450nm.Ifwavelengthsubtractionisavailable,subtractthevaluesof570nmfromthoseof450nmandanalyze data.

6. 7. 8. 9.

Ready-SET-Go Cytokine ELISA Kit Buffers: AssayDiluent(5Xconcentrate):Dilute1/5indIwater. ELISAWashBufferPowder:Reconstitutein1LdIwater SubstrateSolution:Readytouse(1X)100ulperwell. StopSolution:Readytouse(1X)50ulperwell Standard Calibration ThestandardoftheReadySETGo!iscalibratedagainstNIBSCstandards:

TableofStandardCalibration Cytokine hIL2 hIL4 hIL5 hIL6 hIL10 hIL12 hIFNg hTNFa mIL2 mIL4 mIL6 mIFNg* mTNFa ngofeBstandard 1 1 1 1 1 1 1 1 1 1 1 1 1 ngofNIBSCstandard 1.1 2.2 2.2 1.7 0.8 0.8 1.1 0.9 3.1 3 8.5 1.7 UofNIBSCstandard 14.6 22 22 170 4 8 22 36 310 30 850 4.5 340 NIBSCLot# 86/564 88/656 90/586 89/548 93/722 95/544 87/586 87/650 93/566 91/656 93/730 Gg02901533 88/532

*MouseIFNgiscalibratedusingNIHstandard(LotGg02901533)andismeasuredinUnits(U) Table1:TableofStandardCalibration ELISATroubleshootingGuide Problem Possibility Solution 1.Improveefficiencyofwashing.Fillplates completely,soakfor1minuteperwash,asdirected 2.RepeatELISA,becarefulwhenwashingand pipetting 3.Substrateshouldbecolorless

A.HighBackground 1.Improperandinefficientwashing B.Nosignal C.Veryweak signal 2.Crosscontaminationfromotherspecimensorpositive control 3.Contaminatedsubstrate

4.Incorrectdilutions,e.g.,conjugateconcentrationwastoo 4.Repeattestusingcorrectdilutionscheckwiththe high recommendationsoftheantibodymanufacturer 1.Wrongsubstratewasused 1.RepeatELISA,usethecorrectsubstrate

2.Enzymeinhibitorpresentinbufferse.g.,sodiumazide 2.RepeatELISA,makesureyoursystemcontainsno inthewashingbufferandAssayDiluentinhibitsperoxidase enzymeinhibitor activity 1.Improperandinefficientwashing 2.Incorrectdilutionsofstandard 3.Insufficientincubationtime 4.Incorrectstorageofreagents 5.WrongfilterinELISAreaderwasused 1.Makesurewashingprocedureisdonecorrectly 2.Followrecommendationsofstandardhandling exactlyaswrittenonthecertificateofanalysis 3.RepeatELISA,followtheprotocolcarefullyfor eachstepsincubationtime 4.Storereagentsinthecorrecttemperature,avoid freezeandthaw,avoidusingthefrostfreefreezer 5.Usethecorrectwavelengthsetting 1.Makesurewashingprocedureisdonecorrectly seecertificateofanalysis 2.Mixsamplesandreagentsgentlyandequilibrate topropertemperature 3.Platesshouldbewipedonbottombefore measuringabsorbance 4.Donotuseifpastexpirationdate Table2:ELISATroubleshootingGuide

D.Variation 1.Improperandinefficientwashing amongstreplicates 2.Poormixingofsamples 3.Platesnotclean 4.Reagentshaveexpired

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