Deprecated: Required parameter $cat_id follows optional parameter $type in /www/wwwroot/ebimall.com/systems/hong.php on line 2088

Deprecated: Required parameter $where follows optional parameter $tree_id in /www/wwwroot/ebimall.com/systems/hlb.php on line 3505
AntibodiesOnline/P450 Demethylation Activity Kit/ABIN577653/2 x 96 tests188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
您好,欢迎您进入188进口试剂采购网网站! 服务热线:4000-520-616
蚂蚁淘商城 | 现货促销 | 科研狗 | 生物在线

AntibodiesOnline/P450 Demethylation Activity Kit/ABIN577653/2 x 96 tests
Antigen
P450Demethylation
MinimumDetectionLimit
< 100 µU of GST Activity
Application
BiochemicalAssay(BCA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeTheDetectX®P450Activitykitisdesignedtoquantitativelymeasuretheenzymaticactivityofformaldehyde-producingenzymessuchasCytochromeP450s.
BrandDetectX®
DetectionMethodFluorometric
SpecificitySampleTypesvalidated:DemethylatingP450systems:livermicrosomesorcerosomessuchasCypP4503A4,2B4and2D6
CharacteristicsTheP450FluorescentActivitykitallowsactivitymeasurementofanydemethylatingP450systemWITHOUTanyadditionstothetheP450:Substratereaction.ThisassaymeasurestheformaldehydegeneratedbydemethylationandthesignalisreadAFTERtheP450reactionhasbeenterminated.Convenientplateassaywith30minutefluorescentsubstrateincubationanddetectionreadoutat510nm.Testedin3A4,2D6and2B4P450systemswitherythromycin,dextromethorphanorbenzphetamine.ThecytochromeP450s(P450s)areasuperfamilyofhemecontainingenzymesthatdisplaytremendousdiversitywithregardtosubstratespecificityandcatalyticactivity.P450suseaplethoraofbothexogenousandendogenouscompoundsassubstratesintheirreactions.Usuallytheyformpartofmulticomponentelectrontransferreactions.CatalysisbytheeukaryoticP450enzymesinvolvesamultistepreactioncyclethatincludestwostepsinwhichelectrontransferisaccomplishedfromaredoxpartner.Thediflavinprotein,NADPHcytochromeP450reductasecontainsbothFADandFMNandcantransferbothelectronsneededforthecatalyticcycle.InsomeP450reactions,thesecondelectronofthereactioncyclealsocanbedeliveredbycytochromeb5.TheP450enzymesandcofactorsofthemammaliandrug-metabolizingsystemareembeddedinthemembraneoftheendoplasmicreticulum.TheP450splayacrucialroleinthedevelopmentofnewdrugentitiesasdruginteractionscommonlyinhibitcytochromeP450activities.
ComponentsBlackHalfArea96WellPlateTwoplates
AssayBuffer60mLA100mMpotassiumphosphatebufferatpH7.4containing0.005%gentamicin.
NADPHlyophilized2vialsReducedß-nicotinamideadeninedinucleotide2-phosphate(NADPH)freezedriedwithstABIlizersandstoredindesiccators.
StopSolution1mLA1MsolutionofAceticAcidinwater.CAUTION:Acidsolution.
FormaldehydeStandard0.5mL2,000μMformaldehydesolutionindeionizedwater.
Outercontainerhasformaldehydeabsorbingpad.Thestandardisstableifkepttightlysealed.KEEPTIGHTLYSEALED
DetectX®FormaldehydeReagent5mLSpecialformulationofreagentstodetectformaldehydeinsolution.Contains≤0.09%sodiumazideasapreservative.
PlateSealers2each
MaterialnotincludedIncubatorcapableofaccuratelymaintaining37°C.
P450systems.
Microsome,Cerosome,baculosomeorsupersomeP450systems,orrecombinantP450,NADPH/P450oxidoreductaseandcytochromeb5andDilaurylphosphatidylcholine(DLPC)asthelipidusedforreconstitution.
Repeaterpipetwithdisposabletipscapableofdispensing25μL.
Fluorescence96wellplatereadercapableofreADIngfluorescentemissionat510nm,withexcita-tionat450nm.
Setplateparametersfora96-wellCorningCostar3694plate.
See:http://www.
ArborAssays.com/resources/lit.aspforplatedimensiondata.
Thesensitivityoffluorescentassaysisdependantonthecapabilitiesoftheplatereader.
Ifyourplatereaderhasadjustablegainyoucanmodifythesignalsobtainedfromtheassaybyincreas-ingordecreasingthegainsettings,bychangingtheaperturesettingsformonochromatorbasedreaders,orbychangingthebandpasswidthoftheemissionand/orexcitationfiltersonsomereaders.
Pleasereviewtheplatereadermanualfordetails.
SignalsexpressedinthisinsertareRelativeFluorescentUnits(RFU)andwereobtainedonourplatereaders.
TheRFUnumbersyouobtainmaybedifferentfromthese,buttheassayresultsshouldbesimilar.
Softwareforconvertingrawrelativefluorescentunit(FLU)readingsfromtheplatereaderandcarryingoutfourparameterlogisticcurve(4PLC)fitting.
BackgroundThecytochromesP450(P450s)areasuperfamilyofhemecontainingenzymesthatdisplaytre-mendousdiversitywithregardtosubstratespecificityandcat-alyticactivity1,2.P450suseaplethoraofbothexogenousandendogenouscompoundsassubstratesinenzymaticreactions.Usuallytheyformpartofmulticomponentelectrontransferre-actions(seefigure).CatalysisbytheeukaryoticP450enzymesinvolvesamultistepreactioncyclethatincludestwostepsinwhichelectrontransferisaccomplishedfromaredoxpartner.Thediflavinprotein,NADPHcytochromeP450reductase(re-ductase)containsbothFADandFMNandcantransferbothelectronsneededforthecatalyticcycle3.InsomeP450reac-tions,thesecondelectronofthereactioncyclealsocanbede-liveredbycytochromeb54.TheP450enzymesandcofactorsofthemammaliandrug-metabolizingsystemareembeddedinthemembraneoftheendoplasmicreticulum5.TheP450splayacrucialroleinthedevelopmentofnewdrugentitiesasdrug-druginteractionscommonlyarisefromtheinhibitionofcytochromeP450activities.LipidplaysanimportantroleinthereconstitutionofP450-dependentactivitiesafterproteinpu-rification6.MostinvitrostudiesforthereconstitutionofP450activitiesusedilaurylphosphati-dylcholine(DLPC)asthelipidcomponent.ThereconstitutionofenzymaticactivityinvolvesaconcentratedincubationofP450,itsredoxpartners(NADPHandreductase),andlipidfollowedbydilutionintothefinalassaycomponents.Thereportedpreincubationconditionsvarysignifi-cantly7
ApplicationNotesP450enzymesystemsdilutedinthesuppliedAssayBufferprovidedoratypical0.1Mphosphatebufferat pH7.4arecompatIBLewiththisassay.
P450demethylatingreactionconditionsWehaveensuredtheDetectX®P450ActivityAssaydetectstheactivityofthe2B4,2D6and3A4P450systems.
Belowwehavelistedtheconditionsweusedinvalidatingthisfluorescentdetec-tionsystemandtheabilitytoquantitatetheformaldehydeproducedbytheCyp2B4P450enzy-maticreaction.
TypicalCyp2B4EnzymeReactionToduplicatewellsadd15μLofP450enzymesystem(equivalentmolarratiosof2B4P450,Cy-tochromeP450Oxidoreductase,andCytochromeb5inapre-sonicated0.66 mg/mLDLPCsolu-tion),followedby75μLofthesuppliedAssayBufferand5μLofP450substrate.
Sealtheplateandincubatefor5 minutesat37 °Cpriortoadditionof5μLofthereconstitutedsuppliedNADPHactivator.
Sealtheplateagainandincubatefor15 minutesat37 °C.
Add5μLofthesuppliedStopSolutionfollowedbytheadditionof25μLoftheFDRtoeachwell.
Resealtheplateandincubateat37 °Cfor30 minutes.
Forcalibrationpurposestoformaldehyde,the15μLofP450enzymesolu-tionisreplacedwithstandardsmadefromthesuppliedFormaldehydestock.
Forcalibrationpurposestoformaldehyde,theP450enzymesolutionisreplacedwithstandardsmadefromthesuppliedFormaldehydestock.reactionoverviewP450Reaction1.
Carryoutdemethylatingenzymereaction.2.
Stopthereaction(optimal),addFDR.
FormaldehydeDetection3.
Incubateat37 °Cfor30 minutes,readsignal.4.
CalibratetoFormaldehydegenerated.
Plate96wells
ProtocolThekitisuniqueinthatthefluo-rescentsubstrateisnotinvolvedinthemulticomponentP450reaction,butmeasurestheproductofthedemethylation,formaldehyde.
Noseparationorwashingisrequired.
ThekithasbeenvalidatedforseveralP450systemsandshouldworkwithanyBIOLOGicalsystemthatisproducingformaldehydeasaproductofdemethylation.
ThekitprovidesanoptimizedbufferforP450,lyophilizedvialsofthecofactor,NADPH,forthereaction,astableformaldehydestandard,theFormaldehydeDetectionReagent(FDR)andtwo96wellplatesfordetectingthegeneratedfluorescentsignal.
Theenduserwillhavetoprovidethemicrosomal,baculosomesystemortherecombinantP450,reductaseandcytochromeb5systemandanycofactors,etc.necessaryforactivity,alongwithanycandidatedrugs,inhibitorsoractivatorsbeingtested.
ThereactionshouldbecarriedoutinoursuppliedbufferorasimilarPBSbasedbuffersystem.
FollowingtheP450NADPH-inducedreaction,thegenerationofformaldehydecanbestoppedbyadditionofasuitableinhibitor,orthesuppliedstopsolutionofaceticacid.
TheFDRisthenaddedtoallthewells.
Ifcalibrationtoformaldehydeisneeded(forcrosslabcomparisons)thenaformaldehydestandardcurvegeneratedfromthesuppliedstandardshouldberun.
Afterashortincubationat37 °Cfor30 minutes,thefluorescentproductisreadat510nminafluorescentplatereaderwithexcitationat450nm.
TheP450activityisdeterminedbaseduponformaldehydeproduction.
Wehaveprovidedtwo96wellplatesformeasurementbutthisassayisadaptableforhigherdensityplateformats.
Ifsubstitutingtheirownplates,theendusershouldensurethattheirblackHTSplateissuitableforusewiththesereagentspriortorunningsamples.
ReagentPreparation

Allowthekitreagentstocometoroomtemperaturefor30minutes.
Werecommendthatallstan-dardsandsamplesberuninduplicatetoallowtheendusertoaccuratelydetermineP450activity.
EnsurethatallsampleshavereachedoptimaltemperaturefortheP450reactionandhavebeendilutedasappropriatepriortorunningtheminthekit.
NADPHPreparationRemoveavialofNADPHfromthedesiccatorandadd600μLoftheAssayBuffertothevialandvortexthoroughly.
StoreanyunusedreconstitutedNADPHat≤-20°Cfornomorethan2weeks.
FormaldehydeStandardPreparationLabelsixglasstesttubesas#1through#6.
Pipet400μLofAssayBufferintotube#1and250μLintotubes#2-#6.
Add100μLoftheFormaldehydestocksolutiontotube#1andvortexcom-pletely.
Add250μLoftube#1totube#2andvortexcompletely.
Repeattheserialdilutionsfortubes#3through#6.
Theconcentrationofformaldehydeintubes1through6willbe400,200,100,50,25,and12.5μM.
UseallStandardswithin2hourofpreparation.
Std1Std2Std3Std4Std5Std6BufferVolume(μL)400250250250250250AdditionStockStd1Std2Std3Std4Std5VolumeofAddition(μL)100250250250250250FinalConc(μM)400200100502512.5

AssayProcedure
  1. P450reactionvolumeshouldbenomorethan100μLineachwellincludingallcofactors,inhibitorsandactivatorssothat25μLofFDRcanbeaddedtoeachwellfordetection.
    2.Usetheplatelayoutsheetonthebackpageoftheinserttoaidinpropersampleandstandardidentification.P450Reaction
    3.Pipet95μLofAssayBufferasaZerostandard,standardsorsamplesincludingallcofactors,substratesand/orinhibitorsintotheduplicatewellsintheblackplate.Sealwiththeplatesealerandincubatefor15 minutesat37 °C.
    4.Add5μLofthereconstitutedNADPHtoeachwell,sealtheplateandincubateat37 °Cfor15-60 minutes(incubationtimevariesandisbaseduponthesystemandmicrosomesused-seepages7and13).
    5.Add5μLofStopSolutiontoeachwell.FormaldehydeDetection
    6.Add25μLoftheDetectX®FormaldehydeDetectionReagenttoeachwellusingarepeaterpipet.
    7.Gentlytapthesidesoftheplatetoensureadequatemixingofthereagents.
    8.Incubateat37 °Cfor30 minutes.Roomtemperatureincubationwillyieldapproximately75 %ofthefluorescentsignalgeneratedwith37 °Cincubation.
    9.Setplateparametersfora96-wellCorningCostar3694plate.See:http://www.ArborAssays.com/resources/lit.aspforplatedimensiondata.Readthefluorescentsignalfromeachwellinaplatereadercapableofreadingthefluorescentsignalat510nmwithexcitationat450nm.Pleasecontactyourplatereadermanufacturerforsuitablefiltersets.Thisassayrequiresaplatereaderwithefficientfluorescenceoptics.Pleaserefertopage6fordetailsonincreasingsensitivity.
CalculationofResults

AveragetheduplicateFLUreadingsforeachstandardandsample.
Createastandardcurvebyreducingthedatausingthe4PLCfittingroutineontheplatereader,aftersubtractingthemeanFLUsforthezerostandard.
Thesampleactivityobtainedshouldbemultipliedbythedilutionfac-tortoobtainneatsamplevalues.

RestrictionsForResearchUseonly
PrecautionofUseAswithallsuchproducts,thiskitshouldonlybeusedbyqualifiedpersonnelwhohavehadlabo-ratorysafetyinstruction.
Thecompleteinsertshouldbereadandunderstoodbeforeattemptingtousetheproduct.
Someofthecomponentsofthiskitcontainsodiumazideasapreservative,whichmayreactwithleadorcopperplumbingtoformpotentiallyexplosivecomplexes.
Whendisposingofreagentsalwaysflushwithlargevolumesofwatertopreventazidebuild-up.
Storage4°C,RT
StorageCommentAllcomponentsofthiskitshouldbestoredat4°Cuntiltheexpirationdateofthekit.
SupplierImages
image for P450 Demethylation Activity Kit (ABIN577653)P450DemethylationActivityKit
image for P450 Demethylation Activity Kit (ABIN577653)P450DemethylationActivityKit(Image2)
Productcitedin:Talbot,Caperna,Garrett:"GrowthandDevelopmentSymposium:Development,characterization,anduseofaporcineepiblast-derivedliverstemcellline:ARS-PICM-19."in:Journalofanimalscience,Vol.91,Issue1,pp.66-77,2013(PubMed).


新闻动态
行业前沿
技术文章
最新产品

188进口试剂采购网 www.188bio.cn -中国试剂网,试剂网,化学试剂网,国药试剂,抗体公司,试剂公司,试剂盒公司,苏州试剂公司,北京化学试剂公司,天津化学试剂,试剂商城,试剂代理,流式抗体 细胞库查询 sitemap