小鼠cmyc癌基因產(chǎn)物（cmyc）英文說明書

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Mousec-myc

FORRESEARCHUSEONLY

Assayrange：60pg/ml-1600pg/ml96determinations

Purpose

Thiskitallowsforthedeterminationofc-mycconcentrationsinMouseserum,cellculturesupernatesandotherbiologicalfluids

Principleoftheassay

ThekitassayMousec-myclevelinthesample,usePurifiedMousec-mycantibodytocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddc-myctowells,Combinedc-mycantibodywhichWithHRPlabeled,becomeantibody-antigen-enzyme-antibodycomplex,afterwashingCompletely,AddTMBsubstratesolution,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.TheconcentrationofMousec-mycinthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.

Materialsprovidedwiththekit

1

washsolution

20ml×1bottle

7

StopSolution

6ml×1bottle

2

HRP-Conjugatereagent

6ml×1bottle

8

Standard（3200pg/ml）

0.5ml×1bottle

3

Microelisastripplate

12well×8strips

9

Standarddiluent

1.5ml×1bottle

4

Samplediluent

6ml×1bottle

10

Instruction

1

5

ChromogenSolutionA

6ml×1bottle

11

Closureplatemembrane

2

6

ChromogenSolutionB

6ml×1bottle

12

Sealedbags

1

Specimenrequirements

extractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan’t,specimencanbekeptin-20℃topreserve,Avoidrepeatedfreeze-thawcycles.

Can’tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.

Assayprocedure

Diluteandaddsample:DiluteOriginaldensityStandardasfollowtable:

1600pg/ml

5Standard

150μlOriginaldensityStandard+150μlStandarddiluent

800pg/ml

4Standard

150μl5Standard+150μlStandarddiluent

400pg/ml

3Standard

150μl4Standard+150μlStandarddiluent

200pg/ml

2Standard

150μl3Standard+150μlStandarddiluent

100pg/ml

1Standard

150μl2Standard+150μlStandarddiluent

2.Addsample:Setblankwellsseparately(blankcomparisonwellsdon’taddsampleandHRP-Conjugatereagent,othereachstepoperationissame).testingsamplewell.addSampledilution40μltotestingsamplewell,thenaddtestingsample10μl(samplefinaldilutionis5-fold),addsampletowells,don’ttouchthewellwallasfaraspossible,andGentlymix.

3.Incubate:AfterclosingplatewithClosureplatemembrane,incubatefor30minat37℃.

4.Configurateliquid:30-fold(or20-fold)washsolutiondiluted30-fold(or20-fold)withdistilledwaterandreserve.

5.Washing:UncoverClosureplatemembrane,discardLiquid,drybyswing,addwashingbuffertoeverywell,stillfor30sthendrain,repeat5times,drybypat.

6.Addenzyme:AddHRP-Conjugatereagent50μltoeachwell,exceptblankwell.

7.Incubate:Operationwith3.

8.Washing:Operationwith5.

9.Color:AddChromogenSolutionA50ulandChromogenSolutionB50ultoeachwell,evadethelightpreservationfor10minat37℃

10.Stopthereaction:AddStopSolution50μltoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor).

11.Assay:takeblankwellaszero,Readabsorbanceat450nmafterAddingStopSolutionandwithin15min.

Stepsdescription

Standard,Samplediluent

AddStandard,Samplediluent,incubatefor30minat37℃.

Wash5time,AddHRP-Conjugatereagent,incubatefor30minat37℃.

Wash5times,AddChromogenSolutionAandB,incubatefor10minat37℃.

AddStopSolution

Readabsorbanceat450nmwithin15min

calculate

Calculate

Takethestandarddensityasthehorizontal,theODvalueforthevertical,drawthestandardcurveongraphpaper,FindoutthecorrespondingdensityaccordingtothesampleODvaluebytheSamplecurve,multipliedbythedilutionmultiple,orcalculatethestraightlineregressionequationofthestandardcurvewiththestandarddensityandtheODvalue,withthesampleODvalueintheequation,calculatethesampledensity,multipliedbythedilutionfactor,theresultisthesampleactualdensity.

Importantnotes

Thekittakesoutfromtherefrigerationenvironmentshouldbebalanced15-30minutesintheroomtemperature,ELISAplatescoatedifhasnotuseupafteropened,theplateshouldbestoredinSealedbag.

washingbufferwillCrystallizationseparation,itcanbeheatedthewaterhelpsdissolvewhendilute.Washingdoesnotaffecttheresult.

addSamplewithsamplerEachstep,Andproofreaditsaccuracyfrequently,avoidstheexperimentalerror.addsamplewithin5min,ifthenumberofsampleismuch,recommendtouseVolley.

ifthetestingmaterialcontentisexcessivelyhigher(ThesampleODisbiggerthanthefirststandardwell),pleasediluteSample(n-fold),Pleasediluenteandmultipliedbythedilutionfactor.（×n×5）.

Closureplatemembraneonlylimitsthedisposableuse,toavoidcross-contamination.

Thesubstrateevadethelightpreservation.

Pleaseaccordingtouseinstructionstrictly,Thetestresultdetermi