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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Fluorimetric Alanine Aminotransferase Assay Kit/13802/200 Tests
产品编号:13802
市  场 价:¥85560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$4278.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
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AAT Bioquest/Amplite™ Fluorimetric Alanine Aminotransferase Assay Kit/13802/200 Tests
商品介绍
Overview
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Ex/Em(nm)571/585
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryEnzymeDetection
Transferases
RelatedBiochemicalAssays
Alanineaminotransferase(ALT),alsocalledserumglutamatepyruvictransaminase(GPT),isamemberoftransferasefamily.ItcatalyzesthereversIBLetransferofanalpha-aminogroupbetweenalanineandglutamate,andisanimportantenzymeinaminoacidmetabolism.ALTisfoundmainlyinliverandsmallamountinheart,muscle,andkidneys.Inhealthysubjects,serumALTlevelsarelow.However,whencellsaredamaged,suchasacuteandchronichepatitis,obstructivejaundice,carcinomaofliver,myocardialinfarction,ALTmayleakintothebloodstreamandtheALTlevelsaresignificantlyelevated.Therefore,determinationofserumALTlevelhasgreatclinicalanddiagnosticsignificance.Amplite™FluorimetricAlanineAminotransferaseAssayKitprovidesaquickandsensitivemethodforthemeasurementofALTinvariousBIOLOGicalsamples.ALTcatalyzesthereactionofalanineand?-ketoglutaratetopyruvateandglutamate.Theproductglutamateismeasuredbythegenerationofaredfluorescentproductthroughanenzymecoupledreactioncycle.ThesignalcanbereadbyafluorescencemicroplatereaderatEx/Em=530-570nm/590-600nm(optimalEx/Em=540nm/590nm).WiththeAmplite™FluorimetricAlanineAminotransferaseAssayKitaslittleas4mU/mLALTwasdetectedina100µLreactionvolume.Theassayisrobust,andcanbereADIlyadaptedforawidevarietyofapplications.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareserialdilutionsofALTstandard(1to1000mU/mL):

 

1.1   Add100µLDPBSintothevialofALTPositiveControl(ComponentD)tomake100U/mLALTstocksolution.

Note:TheunusedALTstocksolutionshouldbedividedintosingleusealiquotsandstoredat‑20 oC.

1.2   Add10μLof100U/mLALTstandardsolution(fromStep1.1)into990µLDPBSbufferwith0.1%BSAtogenerate1U/mLALTstandardsolution.

1.3   Take300μLof1U/mLALTstandardsolutiontoperform1:3serialdilutionstoget300,100,30,10,3,1and0mU/mLserialdilutionsofALTstandard.

 

1.4   AddserialdilutionsofALTstandardandALTcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.

Note:DilutethetestsamplestotheconcentrationrangeinDPBSbufferwith0.1%BSAifneeded.

 

Table1LayoutofALTstandardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

ALT1

ALT1

….

….

….

….

 

 

 

 

 

 

ALT2

ALT2

 

 

 

 

 

 

 

 

 

 

ALT3

ALT3

 

 

 

 

 

 

 

 

 

 

ALT4

ALT4

 

 

 

 

 

 

 

 

 

 

ALT5

ALT5

 

 

 

 

 

 

 

 

 

 

ALT6

ALT6

 

 

 

 

 

 

 

 

 

 

ALT7

ALT7

 

 

 

 

 

 

 

 

 

 

Note:ALT=ALTStandards,BL=BlankControl,TS=TestSamples.

 

Table2Reagentcompositionforeachwell

ALTStandard

BlankControl

TestSample

SerialDilutions*:50μL

DPBSwith0.1%BSA:50μL

50μL

Note:AddtheseriallydilutedALTstandardsfrom1mU/mLto1U/mLintowellsfromALT1toALT7induplicate.

 

2.PrepareALTassaymixture:

2.1Add100μLofddH2OintothevialofNAD(ComponentC)tohave100XNADsolution.

 

2.2Add10mLofALTAssayBuffer(ComponentB)intothebottleofALTEnzymeMixture

 (ComponentA),andmixwell.

 

2.3Addwholevialof100XNADsolution(fromStep2.1)intotheALTEnzymeMixturesolution(fromStep

 2.2)tohaveALTassaymixture.

Note1:ThisALTassaymixtureisenoughfortwo96-wellplates.Itisunstableatroomtemperature,andshouldbeusedpromptlywithin2hoursandavoidexposuretolight.

Note2:Alternatively,onecanmakea50XofALT EnzymeMixturestocksolutionbyadding200μLofH2OintothebottleofComponentA,andthenpreparetheALTassaymixturebymixthestocksolutionwithassaybuffer(ComponentB)and100xNADsolutionproportionally. Aliquotandstoretheunused50XALTEnzymeMixturestocksolutionand100XNADsolutionat-20oC,andavoidfreeze-thawcycles.

 

 

3.RunALTassay:

3.1   Add50μLofALTassaymixture(fromStep2.3)toeachwellofALTstandard,blankcontrol,andtestsamples(seeStep1.4)tomakethetotalALTassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofALTassaymixtureintoeachwell.

 

3.2   Incubatethereactionat37°Cfor30minto60minutes,protectedfromlight.

Note:ThebackgroundofBlankControlincreaseswithtime.

 

3.3   MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalEx/Em=540/590nm,cutoffat570nm).

References&Citations
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1.   TapperEB,CohenEB,PatelK,BaconB,GordonS,LawitzE,NelsonD,NasserIA,ChalliesT,AfdhalN.(2012)LevelsofAlanineAminotransferaseConfoundUseofTransientElastographytoDiagnoseFibrosisinPatientswithChronicHCVInfection.ClinGastroenterolHepatol.

2.   KainumaM,FurusyoN,AzumaK,KajiwaraE,TakahashiK,NomuraH,TanabeY,SatohT,MaruyamaT,NakamutaM,KotohK,ShimodaS,HayashiJ.(2012)Pegylatedinterferonalpha-2bplusribavirinforJapanesechronichepatitisCpatientswithnormalalanineaminotransferase.HepatolRes,42,33.

3.   Lopez-SuarezA,GuerreroJM,Elvira-GonzalezJ,Beltran-RoblesM,Canas-HormigoF,Bascunana-QuirellA.(2011)Nonalcoholicfattyliverdiseaseisassociatedwithbloodpressureinhypertensiveandnonhypertensiveindividualsfromthegeneralpopulationwithnormallevelsofalanineaminotransferase.EurJGastroenterolHepatol,23,1011.

4.   WangR,LuQ,FengJ,YinF,QinC,LiuB,LiuY,LiuX.(2012)Coexistenceofnon-alcoholicfattyliverdiseasewithelevatedalanineaminotransferaseisassociatedwithinsulinresistanceinyoungHanmales.Endocrine,41,70.

5.   PoustchiH,GeorgeJ,EsmailiS,Esna-AshariF,ArdalanG,SepanlouSG,AlavianSM.(2011)Genderdifferencesinhealthyrangesforserumalanineaminotransferaselevelsinadolescence.PLoSOne,6,e21178.

6.   SanaiFM,HelmyA,DaleC,Al-AshgarH,ABDoAA,KatadaK,AlManaH,SaadehM,Al-HussainiH,AlQuaizM,HashemA,AlSwatK,BzeiziKI,MarottaPJ.(2011)UpdatedthresholdsforalanineaminotransferasedonotexcludesignificanthistologicaldiseaseinchronichepatitisC.LiverInt,31,1039.

7.   ChenCF,LeeWC,YangHI,ChangHC,JenCL,IloejeUH,SuJ,HsiaoCK,WangLY,YouSL,LuSN,ChenCJ.(2011)ChangesinserumlevelsofHBVDNAandalanineaminotransferasedetermineriskforhepatocellularcarcinoma.Gastroenterology,141,1240.

8.   OzawaE,ABIruS,NagaokaS,YanoK,KomoriA,MigitaK,YatsuhashiH,TauraN,IchikawaT,IshibashiH,NakaoK.(2011)Ferritin/alanineaminotransferaseratioasapossibleMarkerforpredictingtheprognosisofacuteliverinjury.JGastroenterolHepatol,26,1326.

9.   OhHJ,KimTH,SohnYW,KimYS,OhYR,ChoEY,ShimSY,ShinSR,HanAL,YoonSJ,KimHC.(2011)Associationofserumalanineaminotransferaseandgamma-glutamyltransferaselevelswithinthereferencerangewithmetabolicsyndromeandnonalcoholicfattyliverdisease.KoreanJHepatol,17,27.

10.   KumarM,SharmaP,GargH,KumarR,BhatiaV,SarinSK.(2011)Transientelastographicevaluationinadultsubjectswithoutovertliverdisease:influenceofalanineaminotransferaselevels.JGastroenterolHepatol,26,1318.


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