
Overview | ![]() PrinterFriendlyVersion |
Ex/Em(nm) | 540/590 |
MW | N/A |
CAS# | N/A |
Solvent | N/A |
Storage | F/D/L |
Category | SmallMoleculeDetection DiagnosticMolecules |
Related |
- Prepareα-ketoglutarateassaymixture:
- MakeAmplite™Redstocksolution(200X):Add50µLofDMSO(ComponentE)intoAmplite™Red(ComponentA)tomake200Xstocksolution.
- Makeassaymixture:
- Add5mLAssayBuffer(ComponentC)intooneEnzymeMix1bottle(ComponentB1)andmixwell.
- Add100µLofddH2OintooneEnzymeMix2vial(ComponentB2)andmixwell.
- 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.
- Prepareseriallydilutedα-ketoglutaratestandardsandtestsamples:
- Prepareα-ketoglutaratestandards:Add10µLof10mMα-KetoglutarateStandard(ComponentD)into990µLofPBStoget100µMα-ketoglutaratesolution.Perform1:3serialdilutionstogetapproximately30,10,3,1,0.3and0.1µMseriallydilutedα-ketoglutaratestandards.
- Addα-ketoglutaratecontainingsamplesandseriallydilutedα-ketoglutaratestandardsintoa96-wellsolidblackmicroplateaccordingtoTables1and2.
- Runα-ketoglutarateassay:
- Add50µLofassaymixture(fromStep1.2.3)intoeachwellofα-ketoglutaratestandard,blankcontrolandtestsamples(seeStep2.2)tomakethetotalα-ketoglutarateassayvolumeof100µL/well.
Note:Fora384-wellplate,add25µLofsample,25µLofassaymixture(fromStep1.2.3)intoeachwell. - Incubatethereactionmixtureat37°Cfor30–60minutes.
- MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm,cutoff=570nm.
- Add50µLofassaymixture(fromStep1.2.3)intoeachwellofα-ketoglutaratestandard,blankcontrolandtestsamples(seeStep2.2)tomakethetotalα-ketoglutarateassayvolumeof100µL/well.
Table1.Layoutofα-ketoglutaratestandardsandtestsamplesina96-wellsolidblackmicroplate.
BL | BL | TS | TS | ... | ... | ||||||
AKG1 | AKG1 | ... | ... | ... | ... | ||||||
AKG2 | AKG2 | ||||||||||
AKG3 | AKG3 | ||||||||||
AKG4 | AKG4 | ||||||||||
AKG5 | AKG5 | ||||||||||
AKG6 | AKG6 | ||||||||||
AKG7 | AKG7 |
Table2.Reagentcompositionforeachwell
α-KetoglutarateStandard | BlankControl | TestSample |
SerialDilutions:50µL | AssayBuffer:50µL | 50µL |
References&Citations | ![]() PrinterFriendlyVersion |
- 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.
- Martin-MoraD,OrtegaA,Reyes-DariasJA,GarciaV,Lopez-FarfanD,MatillaMA,KrellT.(2016)IdentificationofaChemoreceptorinPseudomonasaeruginosaThatSpecificallyMediatesChemotaxisTowardalpha-Ketoglutarate.FrontMicrobiol,7,1937.
- FernandezE,BolanosJP.(2016)alpha-Ketoglutaratedehydrogenasecomplexmoonlighting:ROSsignallingaddedtothelist:AnEditorialhighlightfor"Reductionsinthemitochondrialenzymealpha-ketoglutaratedehydrogenasecomplexinneurodegenerativedisease-beneficialordetrimental?".JNeuRochem,139,689.
- WangZH,AnY,DuMC,QinTJ,LiuYB,XuHZ,YangLQ.(2016)ClinicalAssessmentofHistidine-Tryptophan-KetoglutarateSolutionandModifiedSt.Thomas"SolutioninPediatricCardiacSurgeryofTetralogyofFallot.ArtifOrgans.
- BayliakMM,LylykMP,ShmihelHV,SorochynskaOM,SemchyshynOI,StoreyJM,StoreyKB,LushchakVI.(2017)Dietaryalpha-ketoglutaratepromoteshigherproteinandlowertriacylglyceridelevelsandinducesoxidativestressinlarvaeandyoungadultsbutnotinmiddle-agedDrosophilamelanogaster.CompBiochemPhysiolAMolIntegrPhysiol,204,28.
- ZhaoJ,PengL,CuiR,GuoX,YanM.(2016)Dimethylalpha-ketoglutaratereducesCCl4-inducedliverfibrosisthroughinhibitionofautophagyinhepaticstellatecells.BiochemBiophysResCommun,481,90.
- HangaskyJA,TaabazuingCY,MartinCB,EronSJ,KnappMJ.(2017)Thefacialtriadinthealpha-ketoglutaratedependentoxygenaseFIH:AroleforstericsinlinkingsubstratebindingtoO2activation.JInorgBiochem,166,26.
- GuoH,SuS,MadzakC,ZhouJ,ChenH,ChenG.(2016)Applyingpathwayengineeringtoenhanceproductionofalpha-ketoglutarateinYarrowialipolytica.ApplMicrobiolBiotechnol,100,9875.
- BayliakMM,ShmihelHV,LylykMP,StoreyKB,LushchakVI.(2016)Alpha-ketoglutaratereducesethanoltoxicityinDrosophilamelanogasterbyenhancingalcoholdehydrogenaseactivityandantioxidantcapacity.Alcohol,55,23.
- ZengW,FangF,LiuS,DuG,ChenJ,ZhouJ.(2016)ComparativegenomicsanalysisofaseriesofYarrowialipolyticaWSH-Z06mutantswithvariedcapacityforalpha-ketoglutarateproduction.JBiotechnol,239,76.
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1.模板提取(一般为RNA):Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定:分光光度计或NanoDrop
3.逆转录:逆转录试剂盒(或者一步法试剂盒),这一步可以用普通PCR做,也可以用水域做。
4.荧光定量PCR试剂:通常有用SYBR Green Mix做的,但是这里建议你用EvaGreen做,灵敏度和平行性都要好于SYBR Green,并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他:除了以上的那些还需要离心管、PCR管或板(Axygen反应比较好)、移液枪等,暂时就想到这么多。
1.模板提取(一般为RNA):Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定:分光光度计或NanoDrop
3.逆转录:逆转录试剂盒(或者一步法试剂盒),这一步可以用普通PCR做,也可以用水域做。
4.荧光定量PCR试剂:通常有用SYBR Green Mix做的,但是这里建议你用EvaGreen做,灵敏度和平行性都要好于SYBR Green,并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他:除了以上的那些还需要离心管、PCR管或板(Axygen反应比较好)、移液枪等,暂时就想到这么多。
MicroBCA试剂测定范围是0.5-20μg/ml
楼主自己看看经销商价格
临床实验室对商品定量试剂盒分析性能的验证.pdf(17431.6k)
如果要是直接注射,就不知道了

