DrugNames
GenericName：RatP-IKBaELISAKit.
Purpose
ThiskitallowsforthedeterminationofP-IKBaconcentrationsinRatserum,cellculturesupernatantandotherbiologicalfluids.
Principleoftheassay
ThekitassayRatP-IKBalevelinthesample，usePurifiedRatP-IKBaantibodytocoatmicrotiterplatewells,makesolid-phaseantibody,thenaddP-IKBatowells,CombinedP-IKBaantibodywhichWithHRPlabeledgoatanti-mousebecomeantibody-antigen-enzyme-antibodycomplex,afterwashingCompletely,AddTMBsubstratesolution,TMBsubstratebecomesbluecolorAtHRPenzyme-catalyzed,reactionisterminatedbytheadditionofasulphuricacidsolutionandthecolorchangeismeasuredspectrophotometricallyatawavelengthof450nm.TheconcentrationofP-IKBainthesamplesisthendeterminedbycomparingtheO.D.ofthesamplestothestandardcurve.
Materialsprovidedwiththekit
Materialsprovidedwiththekit
48determinations
96determinations
Storage
Usermanual
1
1
Closureplatemembrane
2
2
Sealedbags
1
1
Microelisastripplate
1
1
2-8℃
Standard：1800ng/L
0.5ml×1bottle
0.5ml×1bottle
2-8℃
Standarddiluent
1.5ml×1bottle
1.5ml×1bottle
2-8℃
HRP-Conjugatereagent
3ml×1bottle
6ml×1bottle
2-8℃
Samplediluent
3ml×1bottle
6ml×1bottle
2-8℃
ChromogenSolutionA
3ml×1bottle
6ml×1bottle
2-8℃
ChromogenSolutionB
3ml×1bottle
6ml×1bottle
2-8℃
StopSolution
3ml×1bottle
6ml×1bottle
2-8℃
washsolution
（20ml×20fold）
×1bottle
（20ml×30fold）
×1bottle
2-8℃
Specimenrequirements
1.serum-coagulationatroomtemperature10-20mins，centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.
2.plasma-usesuitedEDTAorcitrateplasmaasananticoagulant,mix10-20mins,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.
3.Urine-collectsueasterilecontainer,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.TheOperationofHydrothoraxandcerebrospinalfluidReferencetoit.
4.cellculturesupernatant-detectsecretorycomponents,collectsueasterilecontainer,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,detectthecompositionofcells,DilutcellsuspensionwithPBS（PH7.2-7.4）,Cellconcentrationreached1million/ml,repeatedfreeze-thawcycles,damagecellsandreleaseofintracellularcomponents,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant,Ifprecipitationappeared,Centrifugalagain.
5.Tissuesamples-Aftercuttingsamples,checktheweight,addPBS（PH7.2-7.4）,Rapidlyfrozenwithliquidnitrogen,maintainsamplesat2-8℃aftermelting,addPBS（PH7.4）,HomogenizedbyhandorGrinders,centrifugation20-minatthespeedof2000-3000r.p.m.removesupernatant.
6.extractassoonaspossibleafterSpecimencollection,andaccordingtotherelevantliterature,andshouldbeexperimentassoonaspossibleaftertheextraction.Ifitcan’t,specimencanbekeptin-20℃topreserve,Avoidrepeatedfreeze-thawcycles.
7.Can’tdetectthesamplewhichcontainNaN3,becauseNaN3inhibitsHRPactive.
Assayprocedure
1.DiluteandaddsampletoStandard:set10StandardwellsontheELISAplatescoated,addStandard100μltothefirstandthesecondwell,thenaddStandarddilution50μltothefirstandthesecondwell,mix;takeout100μlformthefirstandthesecondwellthenaddittothethirdandtheforthwellseparately.thenaddStandarddilution50μltothethirdandtheforthwell,mix;thentakeout50μlfromthethirdandtheforthwelldiscard,add50μltothefifthandthesixthwell,thenaddStandarddilution50μltothefifthandthesixthwell,mix;takeout50μlfromthefifthandthesixthwellandaddtotheseventhandtheeighthwell,thenaddStandarddilution50μltotheseventhandtheeighthwell,mix;takeout50μlfromtheseventhandtheeighthwellandaddtotheninthandthetenthwell,addStandarddilution50μltotheninthandthetenthwell,mix,takeout50μlfromtheninthandthetenthwelldiscard(addSample50μltoeachwellafterDiluting,(density:1200ng/L,800ng/L,400ng/L,200ng/L,100ng/L)
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：AddChromogenSolutionA50ulandChromogenSolutionBtoeachwell,evadethelightpreservationfor15minat37℃
10.Stopthereaction：AddStopSolution50μltoeachwell,Stopthereaction(thebluecolorchangetoyellowcolor).
11.assay：takeblankwellaszero,Readabsorbanceat450nmafterAddingStopSolutionandwithin15min.
Importantnotes
1.Thekittakesoutfromtherefrigerationenvironmentshouldbebalanced15-30minutesintheroomtemperature,ELISAplatescoatedifhasnotuseupafteropened,theplateshouldbestoredinSealedbag.
2.washingbufferwillCrystallizationseparation,itcanbeheatedthewaterhelpsdissolvewhendilute.Washingdoesnotaffecttheresult.
3.addSamplewithsamplerEachstep,Andproofreaditsaccuracyfrequently,avoidstheexperimentalerror.addsamplewithin5mins,ifthenumberofsampleismuch,recommendtouseVolley.
4.ifthetestingmaterialcontentisexcessivelyhigher(ThesampleODisbiggerthanthefirststandardwell),pleasediluteSample(n-fold),Pleasediluenteandmultipliedbythedilutionfactor.（×n×5）.
5.Closureplatemembraneonlylimitsthedisposableuse,toavoidcross-contamination.
6.Thesubstrateevadethelightpreservation.
7.Pleaseaccordingtouseinstructionstrictly,Thetestresultdeterminationmusttakethemicrotiterplatereaderasastandard.
8.Allsamples,washingbufferandeachkindofrejectshouldaccordingtoinfectivematerialprocess.
9.Donotmixreagentswiththosefromotherlots.
Takethestandarddensityasthehorizontal,theODvalueforthevertical,drawthestandardcurveongraphpaper,FindoutthecorrespondingdensityaccordingtothesampleODvaluebytheSamplecurve,multipliedbythedilutionmultiple,orcalculatethestraightlineregressionequationofthestandardcurvewiththestandarddensityandtheODvalue,withthesampleODvalueintheequation,calculatethesampledensity,multipliedbythedilutionfactor,theresultisthesampleactualdensity.
Calculate
Thischartisforreferenceonly
Assayrange
30ng/L-1500ng/L
Storageandvalidity
1．Storage：2-8℃.
2．validity：sixmonths.
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