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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 α-Ketoglutarate Quantitation Kit/10087/200 Tests
产品编号:10087
市  场 价:¥114560.00
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:$5728.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Fluorimetric α-Ketoglutarate Quantitation Kit/10087/200 Tests
商品介绍
Overview
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Ex/Em(nm)540/590
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategorySmallMoleculeDetection
DiagnosticMolecules
Related
Alpha-ketoglutarate(α-ketoglutarate)isakeymoleculeintheKrebscycledeterminingtheoverallrateofthecitricacidcycleoftheorganism.Asaprecursorofglutamateandglutamine,α-ketoglutarateisacentralmetabolicfuelforcellsofthegastrointestinaltractaswell.Itcandecreaseproteincatabolismandincreaseproteinsynthesistoenhancebonetissueformationintheskeletalmusclesandcanbeusedinclinicalapplications.Alpha-ketoglutarateisusedforkidneydisease;intestinalandstomachdisorders,includingbacterialinfections;liverproblems;cataracts;andrecurringyeastinfections.Itisalsousedforimprovingthewaykidneypatientsreceivinghemodialysistreatmentsprocessprotein.AATBioquest"sAmplite™Colorimetricα-KetoglutarateQuantitationKitoffersasensitivecolorimetricassayforquantifyingα-ketoglutarateinBIOLOGicalsamples.Itutilizesanenzymecoupledreactionthatreleaseshydrogenperoxide,whichcanbedetectedbyAmplite™Redinanabsorbancemicroplatereaderat570nm.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
  1. Prepareα-ketoglutarateassaymixture:
    1. MakeAmplite™Redstocksolution(200X):Add50µLofDMSO(ComponentE)intoAmplite™Red(ComponentA)tomake200Xstocksolution.
    2. Makeassaymixture:
      1. Add5mLAssayBuffer(ComponentC)intooneEnzymeMix1bottle(ComponentB1)andmixwell.
      2. Add100µLofddH2OintooneEnzymeMix2vial(ComponentB2)andmixwell.
      3. Transferentirevial(100µL)ofEnzymeMix2(fromStep1.2.2),and25µLof200XAmplite™Redstocksolution(fromStep1.1)intothevialofEnzymeMix1(fromStep1.2.1)andmixwell.
        Note1:The5mLassaymixtureisenoughforone96-wellplate.Itisnotstable,useitpromptly.
        Note2:Storeunused200XAmplite™Redstocksolutionat-20°C,avoidlightandrepeatedfreeze-thawcycles.
  2. Prepareseriallydilutedα-ketoglutaratestandardsandtestsamples:
    1. Prepareα-ketoglutaratestandards:Add10µLof10mMα-KetoglutarateStandard(ComponentD)into990µLofPBStoget100µMα-ketoglutaratesolution.Perform1:3serialdilutionstogetapproximately30,10,3,1,0.3and0.1µMseriallydilutedα-ketoglutaratestandards.
    2. Addα-ketoglutaratecontainingsamplesandseriallydilutedα-ketoglutaratestandardsintoa96-wellsolidblackmicroplateaccordingtoTables1and2.

  3. Table1.Layoutofα-ketoglutaratestandardsandtestsamplesina96-wellsolidblackmicroplate.
    BLBLTSTS......
    AKG1AKG1............
    AKG2AKG2
    AKG3AKG3
    AKG4AKG4
    AKG5AKG5
    AKG6AKG6
    AKG7AKG7
    Note:AKG=α-KetoglutarateStandard,BL=BlankControl(PBS),TS=TestSample.

    Table2.Reagentcompositionforeachwell
    α-KetoglutarateStandardBlankControlTestSample
    SerialDilutions:50µLAssayBuffer:50µL50µL
    Note:Addtheserialdilutionsofα-ketoglutaratestandardsfrom0.14µMto100µMintowellsfromAKG1toAKG7.

  4. Runα-ketoglutarateassay:
    1. Add50µLofassaymixture(fromStep1.2.3)intoeachwellofα-ketoglutaratestandard,blankcontrolandtestsamples(seeStep2.2)tomakethetotalα-ketoglutarateassayvolumeof100µL/well.
      Note:Fora384-wellplate,add25µLofsample,25µLofassaymixture(fromStep1.2.3)intoeachwell.
    2. Incubatethereactionmixtureat37°Cfor30–60minutes.
    3. MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm,cutoff=570nm.
References&Citations
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  1. YoonWH,SandovalH,Nagarkar-JaiswalS,JaiswalM,YamamotoS,HaeltermanNA,PutluriN,PutluriV,SreekumarA,TosT,AksoyA,DontiT,GrahamBH,OhnoM,NishiE,HunterJ,MuznyDM,CarmichaelJ,ShenJ,ArboledaVA,NelsonSF,WanglerMF,KaracaE,LupskiJR,BellenHJ.(2017)LossofNardilysin,aMitochondrialCo-chaperoneforalpha-KetoglutarateDehydrogenase,PromotesmTORC1ActivationandNeurodegeneration.Neuron,93,115.
  2. Martin-MoraD,OrtegaA,Reyes-DariasJA,GarciaV,Lopez-FarfanD,MatillaMA,KrellT.(2016)IdentificationofaChemoreceptorinPseudomonasaeruginosaThatSpecificallyMediatesChemotaxisTowardalpha-Ketoglutarate.FrontMicrobiol,7,1937.
  3. FernandezE,BolanosJP.(2016)alpha-Ketoglutaratedehydrogenasecomplexmoonlighting:ROSsignallingaddedtothelist:AnEditorialhighlightfor"Reductionsinthemitochondrialenzymealpha-ketoglutaratedehydrogenasecomplexinneurodegenerativedisease-beneficialordetrimental?".JNeuRochem,139,689.
  4. WangZH,AnY,DuMC,QinTJ,LiuYB,XuHZ,YangLQ.(2016)ClinicalAssessmentofHistidine-Tryptophan-KetoglutarateSolutionandModifiedSt.Thomas"SolutioninPediatricCardiacSurgeryofTetralogyofFallot.ArtifOrgans.
  5. BayliakMM,LylykMP,ShmihelHV,SorochynskaOM,SemchyshynOI,StoreyJM,StoreyKB,LushchakVI.(2017)Dietaryalpha-ketoglutaratepromoteshigherproteinandlowertriacylglyceridelevelsandinducesoxidativestressinlarvaeandyoungadultsbutnotinmiddle-agedDrosophilamelanogaster.CompBiochemPhysiolAMolIntegrPhysiol,204,28.
  6. ZhaoJ,PengL,CuiR,GuoX,YanM.(2016)Dimethylalpha-ketoglutaratereducesCCl4-inducedliverfibrosisthroughinhibitionofautophagyinhepaticstellatecells.BiochemBiophysResCommun,481,90.
  7. HangaskyJA,TaabazuingCY,MartinCB,EronSJ,KnappMJ.(2017)Thefacialtriadinthealpha-ketoglutaratedependentoxygenaseFIH:AroleforstericsinlinkingsubstratebindingtoO2activation.JInorgBiochem,166,26.
  8. GuoH,SuS,MadzakC,ZhouJ,ChenH,ChenG.(2016)Applyingpathwayengineeringtoenhanceproductionofalpha-ketoglutarateinYarrowialipolytica.ApplMicrobiolBiotechnol,100,9875.
  9. BayliakMM,ShmihelHV,LylykMP,StoreyKB,LushchakVI.(2016)Alpha-ketoglutaratereducesethanoltoxicityinDrosophilamelanogasterbyenhancingalcoholdehydrogenaseactivityandantioxidantcapacity.Alcohol,55,23.
  10. ZengW,FangF,LiuS,DuG,ChenJ,ZhouJ.(2016)ComparativegenomicsanalysisofaseriesofYarrowialipolyticaWSH-Z06mutantswithvariedcapacityforalpha-ketoglutarateproduction.JBiotechnol,239,76.

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