Overview

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Ex/Em(nm)	570/None
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	SmallMoleculeDetection 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.Perform1:2serialdilutionstoget50,25,12.5,6.25,3.125and1.563µMseriallydilutedα-ketoglutaratestandards.
   2. Addα-ketoglutaratecontainingsamplesandseriallydilutedα-ketoglutaratestandardsintoa96-wellclearbottommicroplateaccordingtoTables1and2.

Table1.Layoutofα-ketoglutaratestandardsandtestsamplesina96-wellclearbottommicroplate.


BL	BL	TS	TS	...	...
AKG1	AKG1	...	...	...	...
AKG2	AKG2
AKG3	AKG3
AKG4	AKG4
AKG5	AKG5
AKG6	AKG6
AKG7	AKG7

Note:AKG=α-KetoglutarateStandard,BL=BlankControl(PBS),TS=TestSample.

Table2.Reagentcompositionforeachwell

α-KetoglutarateStandard	BlankControl	TestSample
SerialDilutions:50µL	AssayBuffer:50µL	50µL

Note:Addtheserialdilutionsofα-ketoglutaratestandardsfrom1.56µMto100µMintowellsfromAKG1toAKG7.


3. 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°Cfor60–90minutes.
   3. MonitortheabsorbanceincreasewithanabsorbanceplatereaderwithpathcheckonatODof570nm.

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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