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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™ Colorimetric Aspartate Aminotransferase (AST) Assay Kit/13801/200 Tests
产品编号:13801
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
公司分类:
AAT Bioquest/Amplite™ Colorimetric Aspartate Aminotransferase (AST) Assay Kit/13801/200 Tests
商品介绍
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Ex/Em(nm)575/None
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryEnzymeDetection
Transferases
RelatedBiochemicalAssays
Aspartateaminotransferase(AST),alsocalledserumglutamicoxaloacetictransaminase(GOT),isamemberoftransferasefamily.ItcatalyzesthereversIBLetransferofanalpha-aminogroupbetweenaspartateandglutamate,andisanimportantenzymeinaminoacidmetabolism.ASTisfoundinmanybodytissuessuchasliver,heart,muscle,kidneys,brain.Inhealthysubjects,serumASTlevelsarelow.However,whencellsaredamaged,suchasacuteandchronichepatitis,obstructivejaundice,carcinomaofliver,myocardialinfarction,ASTmayleakintothebloodstreamandtheASTlevelsaresignificantlyelevated.Therefore,determinationofserumASTlevelhasgreatclinicalanddiagnosticsignificance.Amplite™ColorimetricAspartateAminotransferase(AST)assaykitprovidesaquickandsensitivemethodforthemeasurementofASTinvariousBIOLOGicalsamples.Aspartatetransaminasecatalyzesthereactionofaspartateandα-ketoglutaratetooxaloacetateandglutamate.TheproductL-glutamateismeasuredbythegenerationofabluecolorproductthroughanenzymecoupledreactioncycle.ThesignalcanbereadbyanabsorbancemicroplatereaderatanabsorbanceratioofA570nmtoA610nm.WiththeAmplite™ColorimetricAspartateAminotransferaseAssayKit,wehavedetectedaslittleas2mU/mLASTina100µLreactionvolume.Theassayisrobust,andcanbereADIlyadaptedforawidevarietyofapplications.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareserialdilutionsofASTstandard:

1.1   Add100µLDPBSBuffertoASTPositiveControl(ComponentD)tomake100U/mLASTstandardsolution.

Note:TheunusedASTPositiveControl(ComponentD)shouldbedividedintosingleusealiquotsandstoredat-20oC.

 

1.2   Add5μLof100U/mLASTstandardsolution(fromStep1.1)into997µLDPBSbufferwith0.1%BSAtogenerate500mU/mLASTstandardsolution.

 

1.3   Take400μLof500mU/mLASTstandardsolutiontoperform1:2serialdilutionstoget250,125,62.5,31.25,15.6,7.8and0mU/mLserialdilutionsofASTstandard.

 

1.4   AddserialdilutionsofASTstandardandASTcontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables1and2.

Note:DilutethetestsamplestotheconcentrationrangeinDPBSbufferwith0.1%BSAifneeded.

 

Table1LayoutofASTstandardsandtestsamplesinawhite/clearbottom96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

AST1

AST1

….

….

….

….

 

 

 

 

 

 

AST2

AST2

 

 

 

 

 

 

 

 

 

 

AST3

AST3

 

 

 

 

 

 

 

 

 

 

AST4

AST4

 

 

 

 

 

 

 

 

 

 

AST5

AST5

 

 

 

 

 

 

 

 

 

 

AST6

AST6

 

 

 

 

 

 

 

 

 

 

AST7

AST7

 

 

 

 

 

 

 

 

 

 

Note:AST=ASTStandards,BL=BlankControl,TS=TestSamples.

 

Table2Reagentcompositionforeachwell

ASTStandard

BlankControl

TestSample

SerialDilutions*:50μL

DPBSwith0.1%BSA:50μL

50μL

Note1:AddtheseriallydilutedASTstandardsfrom7.8mU/mLto500mU/mLintowellsfromAST1toAST7induplicate.

Note2:TheASTstandardsareforpositivecontrolonly,andshouldnotbereliedonasaquantitationstandardforenzymeactivity.

 

2.PrepareASTassaymixture:

2.1Add100μLofddH2OintothevialofNAD(ComponentC)tohave100XNADsolution.

 

2.2Add10mLofASTAssayBuffer(ComponentB)intothebottleofASTEnzymeMixture

 (ComponentA),andmixwell.

 

2.3Addwholevialof100XNADsolution(fromStep2.1)intotheASTEnzymeMixturesolution(fromStep

 2.2)tohaveASTassaymixture.

Note1:ThisASTassaymixtureisenoughfortwo96-wellplates.Itisunstableatroomtemperature,andshouldbeusedpromptlywithin2hoursandavoidexposuretolight.

Note2:Alternatively,onecanmakea50XofAST EnzymeMixturestocksolutionbyadding200μLofH2OintothebottleofComponentA,andthenpreparetheASTassaymixturebymixthestocksolutionwithassaybuffer(ComponentB)and100XNADsolutionproportionally. Aliquotandstoretheunused50XASTEnzymeMixturestocksolutionand100XNADsolutionat-20oC,andavoidfreeze-thawcycles.

 

3.RunASTassay:

3.1   Add50μLofASTassaymixture(fromStep2.3)toeachwellofASTstandard,blankcontrol,andtestsamples(seeStep1.4)tomakethetotalASTassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofASTassaymixtureintoeachwell.

 

3.2   Incubatethereactionat37°Cfor30-120minutes,protectedfromlight.

Note:ThebackgroundofBlankControlincreaseswithtimeandtemperature.

 

3.3   MonitortheabsorbanceincreasewithanabsorbanceplatereaderattheabsorbanceratioofA570nm/A610nm.

References&Citations
CitationExplorer

Invitroandinvivostudyoftheapplicationofvolvoxspherestoco-culturevehiclesinlivertissueengineering
Authors:SiouHanChang,HanHsiangHuang,PeiLeunKang,YuChianWu,Ming-HuangChang,ShyhMingKuo
Journal:ActaBiomaterialia(2017)


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