| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 575/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection Transferases |
| Related | BiochemicalAssays |
1.PrepareserialdilutionsofALTstandard(1to1000mU/mL):
1.1Add100µLDPBSintothevialofALTPositiveControl(ComponentD)tomake100U/mLALTstocksolution.Note:TheunusedALTstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
1.2Add10μLof100U/mLALTstandardsolution(fromStep1.1)into990μLDPBSbufferwith0.1%BSAtogenerate1U/mLALTstandardsolution.
1.3Take400μLof1U/mLALTstandardsolutiontoperform1:2serialdilutionstoget500,250,125,62.5,31.25,15.6,and0mU/mLserialdilutionsofALTstandard.
1.4AddserialdilutionsofALTstandardandALTcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.
Note:DilutethetestsamplestotheconcentrationrangeinDPBSbufferwith0.1%BSAifneeded.
Table1LayoutofALTstandardsandtestsamplesinaclear,white,orblackwithclearbottom96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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ALT1 | ALT1 | …. | …. | …. | …. |
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ALT2 | ALT2 |
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ALT3 | ALT3 |
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ALT4 | ALT4 |
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ALT5 | ALT5 |
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ALT6 | ALT6 |
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ALT7 | ALT7 |
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Note:ALT=ALTStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
ALTStandard | BlankControl | TestSample |
SerialDilutions*:50μL | DPBSwith0.1%BSA:50μL | 50μL |
Note:AddtheseriallydilutedALTstandardsfrom15.6mU/mLto1U/mLintowellsfromALT1toALT7induplicate.
2.PrepareALTassaymixture:
2.1Add100μLofddH2OintothevialofNAD(ComponentC)tohave100XNADsolution.
2.2Add10mLofALTAssayBuffer(ComponentB)intothebottleofALTEnzymeMixture(ComponentA),andmixwell.
2.3Addwholevialof100XNADsolution(fromStep2.1)intotheALTEnzymeMixturesolution(fromStep2.2)tohaveALTassaymixture.
Note1:ThisALTassaymixtureisenoughfortwo96-wellplates.Itisunstableatroomtemperature,andshouldbeusedpromptlywithin2hoursandavoidexposuretolight.
Note2:Alternatively,onecanmakea50XofALTEnzymeMixturestocksolutionbyadding200μLofH2OintothebottleofComponentA,andthenpreparetheALTassaymixturebymixthestocksolutionwithassaybuffer(ComponentB)and100XNADsolutionproportionally.Aliquotandstoretheunused50XALTEnzymeMixturestocksolutionand100XNADsolutionat-20oC,andavoidfreeze-thawcycles.
3.RunALTassay:
3.1Add50μLofALTassaymixture(fromStep2.3)toeachwellofALTstandard,blankcontrol,andtestsamples(seeStep1.4)tomakethetotalALTassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofALTassaymixtureintoeachwell.
3.2Incubatethereactionat37°Cfor60minto120minutes,protectedfromlight.
Note:ThebackgroundofBlankControlincreaseswithtime.
3.3MonitortheabsorbanceincreasewithanabsorbanceplatereaderattheabsorbanceratioofA570nm/A610nm.
| References&Citations |  PrinterFriendlyVersion |
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.
