Mouse （VLDL）ELISA Kit

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Mouseverylowdensitylipoprotein(VLDL)ELISAKitFORRESEARCHUSEONLY.NotforclinicaldiagnosisuseCATALOG#:DAG859INTRODUCTION?ThiskitallowsforthedeterminationofVLDLconcentrationsinMouseserum?Detectionofspecies:Mouse?Detectionmedium:serum,cellculturesupernates.?Assayrange：6.0μg/ml-160μg/mlPRINCIPLEOFTESTThekitassayMouseVLDLlevelinthesample，usePurifiedMouseVLDLantibodytocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddVLDLtowells,CombinedVLDLantibodywhichWithHRPlabeled,becomeantibody-antigen-enzyme-antibodycomplex,afterwashingCompletely,AddTMBsubstratesolution,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.TheconcentrationofMouseVLDLinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.Phone:(612)379-2956Phone:(800)343-7475Fax:(612)656-4400Catalog#:DAG6342COMPOSITIONOFTHEKIT1washsolution20ml×1bottle7StopSolution6ml×1bottle2HRP-Conjugatereagent6ml×1bottle8Standard（320μg/ml）0.5ml×1bottle3Microelisastripplate12well×8strips9Standarddiluent1.5ml×1bottle4Samplediluent6ml×1bottle10Instruction15ChromogenSolutionA6ml×1bottle11Closureplatemembrane26ChromogenSolutionB6ml×1bottle12Sealedbags1STORAGECONDITIONS?Theunopenedkitshallbestoredat[2-8℃].?Foropenedkitcanbestoredat[2-8℃]forupto1month.Ifnotbeusedrecently,thestandardshouldbekeptin-20℃.WASHINGMETHOD?Manuallywashingmethod:shakeawaytheremainedliquidintheenzymeplates;placesomebibulouspapersonthetest-bed,andflaptheplatesontheupsidedownstrongly.Injectatleast0.35mlafter-dilutionwashingsolutionintothewell,andmarinate1~2minutes.Repeatthisprocessaccordingtoyourrequirements.?Automaticwashingmethod:ifthereisautomaticwashingmachine,itshouldonlybeusedinthetestwhenyouarequitefamiliarwithitsfunctionandperformance.SAMPLEPREPARATION1.extractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan’t,specimencanbekeptin-20℃topreserve,Avoidrepeatedfreeze-thawcycles.Phone:(612)379-2956Phone:(800)343-7475Fax:(612)656-4400Catalog#:DAG63432.Can’tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.ASSAYPROCEDUREStep1:Diluteandaddsample:DiluteOriginaldensityStandardasfollowtable:Step2:Setblankwellsseparately(blankcomparisonwellsdon’taddsampleandHRP-Conjugatereagent,othereachstepoperationissame).testingsamplewell.addSampledilution40μltotestingsamplewell,thenaddtestingsample10μl(samplefinaldilutionis5-fold),addsampletowells,don’ttouchthewellwallasfaraspossible,andGentlymix.Step3:Incubate:Coverwiththeadhesivestripprovided,incubatefor30minat37℃.Step4:Configurateliquid:Dilutewashsolution30-fold(or20-fold)withdistilledwater.Step5:Washing:Uncovertheadhesivestrip,discardliquid,Pipettewashingbuffertoeverywell,stillfor30sthendrain,repeat5times.Step6:Addenzyme:PipetteHRP-Conjugatereagent50μltoeachwell,exceptblankwell.Step7:Incubate:Operationwith3.Step8:Washing:Operationwith5.160μg/ml5Standard150μlOriginaldensityStandard+150μlStandarddiluent80μg/ml4Standard150μl5Standard+150μlStandarddiluent40μg/ml3Standard150μl4Standard+150μlStandarddiluent20μg/ml2Standard150μl3Standard+150μlStandarddiluent10μg/ml1Standard150μl2Standard+150μlStandarddiluentPhone:(612)379-2956Phone:(800)343-7475Fax:(612)656-4400Catalog#:DAG6344Step9:Color:PipetteChromogenSolutionA50ulandChromogenSolutionBtoeachwell,avoidthelightpreservationfor15minat37℃Step10:Stopthereaction:PipetteStopSolution50μltoeachwell,Stopthereaction(thebluechangetoyellow).Step11:Calculate:takeblankwellaszero,Readabsorbanceat450nmafterPipetteingStopSolutionwithin15min.CALCULATIONOFRESULTTakethestandarddensityasthehorizontal,theODvalueforthevertical,drawthestandardcurveongraphpaper,FindoutthecorrespondingdensityaccordingtothesampleODvaluebytheSamplecurve,multipliedbythedilutionmultiple,orcalculatethestraightlineregressionequationofthestandardcurvewiththestandarddensityandtheODvalue,withthesampleODvalueintheequation,calculatethesampledensity,multipliedbythedilutionfactor,theresultisthesampleactualdensity.EXPIRATIONSixmonths[seelabelontheouterboxforthespecificdate].Phone:(612)379-2956Phone:(800)343-7475Fax:(612)656-4400Catalog#:DAG6345ATTENTION?Thekittakesoutfromtherefrigerationshouldbebalanced15-30minutesintheroomtemperature,ifthecoatedELISAplateshavenotbeenusedupafteropening,theplateshouldbestoredinsealedbag.?washingbufferwillCrystallizationseparation,itcanbeheatedthewaterhelpsdissolvewhendilute.Washingdoesnotaffecttheresult.?addSamplewithsamplerEachstep,Andproofreaditsaccuracyfrequently,avoidstheexperimentalerror.addsamplewithin5min,ifthenumberofsampleismuch,recommendtouseVolley.?ifthetestingmaterialcontentisexcessivelyhigher(ThesampleODisbiggerthanthefirststandardwell),pleasediluteSample(n-fold),Pleasediluenteandmultipliedbythedilutionfactor.（×n×5）.?Closureplatemembraneonlylimitsthedisposableuse,toavoidcross-contamination.?Thesubstrateshouldevadethelighttobepreserved.?Pleaserefertotheuserinstructionstrictly,thetestresultdeterminationmusttakethemicrotiterplatereaderasastandard.?Thepreparationofsamplesandallthereagentsshouldrefertoinfectivematerialprocess.?Donotmixreagentswiththosefromotherlots
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