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AntibodiesOnline/Human Apoptosis Array C1/ABIN625525/2 samples

PointeScientific经销商 /发布时间:1503723304 /浏览量:14

Reactivity

Human

MethodType

SandwichELISA

Application

AntibodyArray(AA)

Options

Supplier

SupplierProductNo.

Productdetails ReferencesImagesHandlingApplicationDetailsTargetdetailsbacktotop
Productdetails ShowHide
Purpose	C-SeriesHumanApoptosisAntibodyArray1Kit.Detects43HumanApoptoticFactors.Suitableforallliquidsampletypesbutintendedforusewithcellandtissuelysates.
Brand	RayBio®
SampleType	Serum,Plasma,CellCultureSupernatant,CellLysate,TissueLysate
AnalyticalMethod	Semi-Quantitative
DetectionMethod	Chemiluminescent
Specificity	Bad,bax,bcl-2,bcl-w,BID,BIM,Caspase-3,Caspase-8,CD40(TNFRSF5),CD40Ligand(TNFSF5),cIAP-2,CytochromeC,DR6(TNFRSF21),Fas(TNFRSF6/Apo-1),FasLigand(TNFSF6),HSP27,HSP60,HSP70,HTRA2,IGF-1,IGF-2,IGFBP-1,IGFBP-2,IGFBP-3,IGFBP-4,IGFBP-5,IGFBP-6,IGF-1R,livin,p21,p27,p53,SMAC,Survivin(BIRC5),TNFRI(TNFRSF1A),TNFRII(TNFRSF1B),TNFalpha,TNFbeta(TNFSF1B),TRAILR1(TNFRSF10A/DR4),TRAILR2(TNFRSF10B/DR5),TRAILR3(TNFRSF10C),TRAILR4(TNFRSF10D),XIAP
Characteristics	Easytouse Nospecializedequipmentneeded Compatiblewithnearlyanyliquidsample Proventechnology(manypublications) Highlysensitive(pg/mL) SandwichELISAspecificity HigherdensitythanELISA,Westernblotorbead-basedmultiplex
Components	AntibodyArrayMembranes BiotinylatedDetectionAntibodyCocktail BlockingBuffer WashBuffers1and2 Cell&TissueLysisBuffer DetectionBuffersCandD PlasticIncubationTray ProteaseInhibitorCocktail(inselectkits)
Materialnotincluded	Pipettors,pipettipsandothercommonlabconsumables Orbitalshakeroroscillatingrocker TissuePaper,blottingpaperorchromatographypaper AdhesivetapeorSaranWrap Distilledorde-ionizedwater Achemiluminescentblotdocumentationsystem(suchasUVPsChemiDoc-It®orEpiChemIIBenchtopDarkroom),X-rayFilmandasuitablefilmprocessor,oranotherchemiluminescentdetectionsystem.

Targetdetails ReferencesImagesHandlingApplicationDetailsbacktotop
Targetdetails ShowHide
Background	Apoptosisistheprocessofprogrammedcelldeaththatinvolvesaseriesofbiochemicaleventsleadingtocharacteristiccellmorphologyanddeath.Theseeventsincludeblebbing,changestothecellmembranesuchaslossofmembraneasymmetryandattachment,cellshrinkage,nuclearfragmentation,chromatincondensation,andchromosomalDNAfragmentation.Studiesonapoptosishaveincreasedsubstantiallysincetheearly1990s.Inadditiontoitsimportanceasabiologicalphenomenonsuchascelltermination,homeostasis,developmentandlymphocyteinteractions,deregulationofapoptosishasbeenimplicatedinmanydiseases.Excessiveapoptosiscauseshypotrophy,suchasinischemicdamage,whereasinsufficientapoptosisresultsinuncontrolledcellproliferation,suchasHIVprogressionandcancerdevelopment.Apoptosisismediatedbyadiverserangeofcellsignals,bothextracellularandintracellular.Extracellularsignalsmayincludetoxins,hormones,growthfactors,nitricoxideorcytokines.Intracellularapoptoticsignalingmaybeinducedinresponsetostressvia,heat,radiation,nutrientdeprivation,viralinfection,hypoxiaandincreasedintracellularcalciumconcentrationorthebindingofnuclearreceptorsbyglucocorticoids.Thesesignalsmaypositivelyornegativelyinduceapoptosis.Twoapoptoticsignaltransductionpathwaysinmammalshavebeenreported:theTNF-inducedmodelandtheFas-Fasligand-mediatedmodel.TNFisthemajorextrinsicmediatorofapoptosis.MostcellsinthehumanbodyhavetworeceptorsforTNF:TNF-R1andTNF-R2.ThebindingofTNFtoTNF-R1hasbeenshowntoinitiatethepathwaythatleadstocaspaseactivationviatheintermediatemembraneproteinsTNFreceptor-associateddeathdomain(TRADD)andFas-associateddeathdomainprotein(FADD).Bindingofthisreceptorcanalsoindirectlyleadtotheactivationoftranscriptionfactorsinvolvedincellsurvivalandinflammatoryresponses.TheFasreceptor(alsoknownasApo-1orCD95)bindstheFasligand.TheinteractionbetweenFasandFasLresultsintheformationofthedeath-inducingsignalingcomplex(DISC),whichcontainstheFADD,caspase-8andcaspase-10.FollowingTNF-R1andFasactivationinmammaliancellsabalancebetweenpro-apoptotic(BAX,BID,BAK,orBAD)andanti-apoptotic(Bcl-XlandBcl-2)membersoftheBcl-2familyisestablished.Thisbalanceistheproportionofpro-apoptotichomodimersthatformintheouter-membraneofmitochondrion.Thepro-apoptotichomodimersarerequiredtomakethemitochondrialmembranepermeableforthereleaseofcaspaseactivatorssuchascytochromecandSMAC.Controlofpro-apoptoticproteinsundernormalcellconditionsofnon-apoptoticcellsisincompletelyunderstood.Mitochondriaareanimportantsiteforapoptosis.MitochondrialproteinsknownasSMACs(secondmitochondria-derivedactivatorofcaspases)arereleasedintothecytosolfollowinganincreaseinpermeability.SMACbindstoinhibitorofapoptosisproteins(IAPs)anddeactivatesthem,preventingtheIAPsfromarrestingtheapoptoticprocessandthereforeallowingapoptosistoproceed.IAPalsonormallysuppressestheactivityofcaspases(cysteineasparticacidproteases),whichcarryoutthedegradationofthecell.Therefore,thedegradativeactivityofcaspasesseemstobeindirectlyregulatedbymitochondrialpermeability.Cytochromecisalsoreleasedfrommitochondriaduetoformationofachannel,MAC,intheoutermitochondrialmembrane,andservesaregulatoryfunctionasitprecedesmorphologicalchangeassociatedwithapoptosis.OncecytochromecisreleaseditbindswithApaf-1andATP,whichthenbindtopro-caspase-9tocreateaproteincomplexknownasanapoptosome.Theapoptosomecleavesthepro-caspasetoitsactiveformofcaspase-9,whichinturnactivatestheeffectorcaspase-3.Thetumor-suppressorproteinp53alsoplayscriticalroleinapoptosis.p53accumulatesinresponsetoDNAdamageviainterferon-alphaandinterferon-betapathways,whichinducetranscriptionofthep53geneandresultintheincreaseofp53proteinlevelandenhancementofcancercellapoptosis.p53preventsthecellfromreplicatingbystoppingthecellcycleatG1,orinterphase,togivethecelltimetorepair,howeveritwillinduceapoptosisifdamageisextensiveandrepaireffortsfail.Anydisruptiontotheregulationofthep53orinterferongeneswillresultinimpairedapoptosisandthepossibleformationoftumors.ArecentreporthasshowntheinvolvementofIGFBPs(insulin-likegrowthfactor-bindingprotein)inapoptosis.IGFBP1proteinlocalizestomitochondriawhereitbindstotheBAKandhindersBAKactivationandapoptosisinduction.WhenIGFBP1isinacomplexwithBAK,formationofaproapoptoticp53/BAKcomplexandapoptosisinductionisimpaired,bothinculturedcellsandinliver.Incontrast,liversofIGFBP1-deficientmiceexhibitspontaneousapoptosisthatisaccompaniedbyp53mitochondrialaccumulationandBAKoligomerization.TheseresultsidentifyIGFBP1asanegativeregulatoroftheBAK-dependentpathwayofapoptosis,whoseexpressionintegratesthetranscriptionalandmitochondrialfunctionsofthep53tumorsuppressorprotein.

ApplicationDetails ReferencesImagesHandlingTargetdetailsbacktotop
ApplicationDetails ShowHide
ApplicationNotes	PerformALLincubationandwashstepsundergentlerotationorrockingmotion(~0.5to1cycle/sec)usinganorbitalshakeroroscillatingrockertoensurecompleteandevenreagent/samplecoverage.Rocking/rotatingtoovigorouslymaycausefoamingorbubblestoappearonthemembranesurfacewhich,shouldbeavoided.AllwashesandincubationsshouldbeperformedintheIncubationTray(ITEM10)providedinthekit.CovertheIncubationTraywiththelidprovidedduringallincubationstepstoavoidevaporationandoutsidedebriscontamination.Ensurethemembranesarecompletelycoveredwithsufficientsampleorreagentvolumeduringeachincubation.Avoidforcefulpipettingdirectlyontothemembrane,instead,gentlypipettesamplesandreagentsintoacornerofeachwell.Aspiratesamplesandreagentscompletelyaftereachstepbysuctioningoffexcessliquidwithapipette.Tiltingthetraysotheliquidmovestoacornerandthenpipettingisaneffectivemethod.Optionalovernightincubationsmaybeperformedforthefollowingsteptoincreaseoverallspotsignalintensities: -SampleIncubation -BiotinylatedAntibodyCocktailIncubation -HRP-StreptavidinIncubation
Comment	TheC-Seriesarraysfeaturechemiluminescentsignaldetection.TheantibodiesarespottedonnitrocellulosemembranesolidsupportsandarehandledinaverysimilarmannertoWesternblots. AllC-SeriesarraysworkonthesandwichELISAprinciple,utilizingamatchedpairofantibodies:animmobilizedcaptureantibodyandacorrespondingbiotinylateddetectionantibody.
SampleVolume	1mL
Plate	Membrane
Protocol	Blockmembranes IncubatewithSample IncubatewithBiotinylatedDetectionAntibodyCocktail IncubatewithHRP-ConjugatedStreptavidin IncubatewithDetectionBuffers Imagewithchemiluminescentimagingsystem Performdensitometryandanalysis
SamplePreparation	Useserum-freeconditionedmediaifpossible.Ifserum-containingconditionedmediaisrequired,itishighlyrecommendedthatcompletemediumbeusedasacontrolsincemanytypesofseracontainscytokines.Werecommendthefollowingparametersforyoursamples:50to100µloforiginalordilutedserum,plasma,cellculturemedia,orotherbodyfluid,or50-500µg/mlofproteinforcellandtissuelysates.Ifyouexperiencehighbackgroundorifthefluorescentsignalintensitiesexceedthedetectionrange,furtherdilutionofyoursampleisrecommended.
AssayProcedure	Placeeachmembraneintotheprovidedeight-welltray(-meanstheantibodyprintedside).2.Add2ml1XBlockingBufferandincubateatroomtemperaturefor30mintoblockmembranes.Note:incubationmaybedoneat4°Cforovernight.3.Incubatemembraneswith1mlofsampleatroomtemperaturefor1to2hours.Dilutesampleusing1XBlockingBufferifnecessary.Note:Werecommendusing1mlofConditionedmediaor1mloforiginalor10-folddilutedseraorplasmaor50-500µgofproteinforcelllysatesandtissuelysates.Dilutethelysateatleast10foldswith1Xblockingbuffer.Note:Theamountofsampleuseddependsontheabundanceofcytokines.Moreofthesamplecanbeusedifthesignalsaretooweak.Ifthesignalsaretoostrong,thesamplecanbedilutedfurther.Note:Incubationmaybedoneat4°Cforovernight.4.Decantthesamplesfromeachcontainer,andwash3timeswith2mlof1XWashBufferIatroomtemperaturewithshaking.Pleaseallow5minperwash.Dilute20XWashBufferIwithH2O.5.Wash2timeswith2mlof1XWashBufferIIatroomtemperaturewithshaking.Allow5minperwash.Dilute20XWashBufferIIwithH2O.6.Prepareworkingsolutionforprimaryantibody.Add100µlof1XblockingbuffertotheBiotin-ConjugatedAnti-Cytokinestube.Mixgentlyandtransferallmixturetoatubecontaining2mlof1Xblockingbuffer.Note:thedilutedbiotin-conjugatedantibodiescanbestoredat4°Cfor2-3days.7.Add1mlofdilutedbiotin-conjugatedantibodiestoeachmembrane.Incubateatroomtemperaturefor1-2hours.Note:incubationmaybedoneat4°Cforovernight.8.Washasdirectedinsteps4and5.9.Add2mlof1,000folddilutedHRP-conjugatedstreptavidin(e.g.add2µlofHRP-conjugatedstreptavidinto1998µl1XBlockingBuffer)toeachmembrane.Note:Mixthetubecontaining1,000XHRP-ConjugatedStreptavidinwellbeforeusesinceprecipitationmayformduringstorage.10.Incubateatroomtemperaturefor2hours.Note:incubationmaybedoneat4°Cforovernight.11.Washasdirectedinsteps4and5. Donotletthemembranedryoutduringdetection.Thedetectionprocessmustbecompletedwithin40minuteswithoutstopping.1.Proceedwiththedetectionreaction.Add250µlof1XDetectionBufferCand250µlof1XDetectionBufferDforonemembrane,mixbothsolutions.Drainoffexcesswashbufferbyholdingthemembraneverticallywithforceps.Placemembraneproteinsideup(-markisontheproteinsidetopleftcorner)onacleanplasticsheet(providedinthekit).PipettethemixedDetectionBufferontothemembraneandincubateatroomtemperaturefor2minutes.Ensurethatthedetectionmixtureiscompletelyandevenlycoveringthemembranewithoutanyairbubbles.2.Drainoffanyexcessdetectionreagentbyholdingthemembraneverticallywithforcepsandtouchingtheedgeagainstatissue.Gentlyplacethemembrane,proteinsideup,onapieceofplasticsheet(-markisontheproteinsidetopleftcorner).Coverwithanotherpieceofplasticsheetonthearray.Gentlysmoothoutanyairbubbles.Avoidusingpressureonthemembrane.3.Exposethearraytox-rayfilm(werecommendtouseKodakx-omatARfilm)anddetectsignalusingfilmdeveloper.Orthesignalcanbedetecteddirectlyfromthemembraneusingachemiluminescenceimagingsystem.Exposethemembranesfor40secondsandthenre-exposethefilmaccordingtotheintensityofsignals.Ifthesignalsaretoostrong(backgroundtoohigh),reduceexposuretime(e.g.5-30seconds).Ifthesignalsaretooweak,increaseexposuretime(e.g.5-20minorovernight).Orre-incubatemembranesovernightwith1xHRP-conjugatedstreptavidin,andredodetectioninthesecondday.4.Savemembranesin-20°Cto-80°Cforfuturereference.
CalculationofResults	Visualcomparisonofarrayimagesmaybesufficienttoseedifferencesinrelativeproteinexpression.However,mostresearcherswillwanttoperformnumericalcomparisonsofthesignalintensities(ormoreprecisely,signaldensities),using2-Ddensitometry.Gel/Blotdocumentationsystemsandotherchemiluminescentorphosphorescentdetectionsystemsareusuallysoldasapackagewithcompatibledensitometrysoftware.Anydensitometrysoftwareshouldbesufficienttoobtainspotsignaldensitiesfromyourscannedimages.Onesuchsoftwareprogram,ImageJ,isavailableforfreefromtheNIHwebsitealongwithanarrayplug-in.
AssayPrecision	Inter-arrayCoefficientofVariation(CV)ofspotsignalintensitiesaslowas5%whenrununderoptimalconditions.
Restrictions	ForResearchUseonly

Handling ReferencesImagesApplicationDetailsTargetdetailsbacktotop
Handling ShowHide
HandlingAdvice	Theantibodyprintedsideofeachmembraneismarkedbyadash(-)ornumber(#)intheupperleftcorner.DonotallowmembranestodryoutduringtheexperimentortheymaybecomefragileandbreakORhighand/orunevenbackgroundmayoccur.Graspmembranesbythecornersoredgesonlyusingforceps.DONOTtouchprintedantibodyspots.
Storage	-20°C
StorageComment	Forbestresults,storetheentirekitfrozenat-20°Cuponarrival.Storedfrozen,thekitwillbestableforatleast6monthswhichisthedurationoftheproductwarrantyperiod.Oncethawed,storearraymembranesat-20°Candallotherreagentsundilutedat4°Cfornomorethan3months.
ExpiryDate	6months

Images ReferencesHandlingApplicationDetailsTargetdetailsbacktotop
Images ShowHide
SupplierImages	HumanApoptosisArrayC1

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