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AAT Bioquest/Amplite™ Fluorimetric Aspartate Aminotransferase (AST) Assay Kit/13800/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection Transferases |
| Related | BiochemicalAssays |
1.PrepareserialdilutionsofASTstandard(1to300mU/mL):
1.1 Add100µLDPBSBuffertoASTPositiveControl(ComponentD)tomake100U/mLASTstandardsolution.
Note:TheunusedASTPositiveControl(ComponentD)shouldbedividedintosinglueusealiquotsandstoredat-20oC.
1.2 Add3μLof100U/mLASTstandardsolution(fromStep1.1)into997µLDPBSbufferwith0.1%BSAtogenerate300mU/mLASTstandardsolution.
1.3 Take300μLof300mU/mLASTstandardsolutiontoperform1:3serialdilutionstoget100,30,10,3,1,0.3and0mU/mLserialdilutionsofASTstandard.
1.4 AddserialdilutionsofASTstandardandASTcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Note:DilutethetestsamplestotheconcentrationrangeinDPBSbufferwith0.1%BSAifneeded.
Table1LayoutofASTstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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AST1 | AST1 | …. | …. | …. | …. |
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AST2 | AST2 |
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AST3 | AST3 |
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AST4 | AST4 |
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AST5 | AST5 |
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AST6 | AST6 |
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AST7 | AST7 |
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Note:AST=ASTStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
ASTStandard | BlankControl | TestSample |
SerialDilutions*:50μL | DPBSwith0.1%BSA:50μL | 50μL |
Note1:AddtheseriallydilutedASTstandardsfrom0.3mU/mLto300mU/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,onecanmakea50XofASTEnzymeMixturestocksolutionbyadding200μLofH2OintothebottleofComponentA,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°Cfor20-30minutesorroomtemperaturefor60minutes,protectedfromlight.
Note:ThebackgroundofBlankControlincreaseswithtimeandtemperature.
3.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalEx/Em=540/590nm,cutoffat570nm).
| References&Citations |  CitationExplorer |
Invitroandinvivostudyoftheapplicationofvolvoxspherestoco-culturevehiclesinlivertissueengineering
Authors:SiouHanChang,HanHsiangHuang,PeiLeunKang,YuChianWu,Ming-HuangChang,ShyhMingKuo
Journal:ActaBiomaterialia(2017)
