| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 575/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
1.Preparestocksolutions:
1.1 Amplite™Redsubstratestocksolution(200X):Add50µLofDMSO(ComponentE)intothevialofAmplite™Redsubstrate(ComponentA)tomakea200Xstocksolution.
Note:TheunusedAmplite™Redsubstratestocksolutionshouldbedividedintosingleusealiquots.Storeat-20oCandavoidexposuretolight.
1.2 G3Pstocksolution:Add250µLofddH2OintothevialofG3PStandard(ComponentD)tomake10mMG3Pstocksolution.
Note:TheunusedG3Pstocksolutionshouldbedividedintosingleusealiquotsandstoredat‑20 oC.
2.PrepareGlycerol3-Phosphate(G3P)assaymixture:
2.1 Add5mLofAssayBuffer(ComponentC)tothebottleofEnzymeMix(ComponentB)andmixwell.
2.2 Add25µLofAmplite™Redsubstratestocksolution(200X,fromStep1.1)intothebottleofComponentsBandC(fromStep2.1)tomaketheG3Passaymixture(ComponentsA,BandC).
Note1:TheG3Passaymixtureshouldbeusedpromptlyandkeptfromlight.
Note2:OnecandividetheunusedmixtureofComponentsBandCintosingleusealiquotsandstoredat-20ºC.
3.PrepareserialdilutionsofGlycerol3-Phosphatestandard(0to200μM):
3.1 Add100μLof10mMG3Pstandardstocksolution(fromStep1.2)to900µL1×PBSbuffertogenerate1000μMG3Pstandardsolution.
Note:DilutedG3Pstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof1000μMG3Pstandard(fromStep3.1)to800µL1×PBSbuffertogenerate200μMG3Pstandardsolution,andthenperform1:2serialdilutionstoget200,100,50,25,12.5,6.25,and0μMG3Pstandard.
3.3 AddG3Pstandardsandtestsamplesintoa96-wellclearbottom/blackwallmicroplateasdescribedinTables1and2.
Note:Treatthecellsortissuesamplesasdesired.
Table1.LayoutofG3Pstandardsandtestsamplesinaclearbottom96-wellmicroplate*
BL | BL | TS | TS | …. | …. |
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CS1 | CS1 | …. | …. | …. | …. |
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CS2 | CS2 |
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CS3 | CS3 |
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CS4 | CS4 |
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CS5 | CS5 |
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CS6 | CS6 |
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CS7 | CS7 |
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*Note:CS=G3PStandards;BL=BlankControl;TS=TestSamples
Table2.Reagentcompositionforeachwell*
G3PStandard | BlankControl | TestSample |
Serialdilutions*(50μL) | Assaybuffer:50μL | 50μL |
*Note:AddtheseriallydilutionsofG3Pstandardsfrom6.25to1000μMintowellsfromCS1toCSinduplicate.
4.RunGlycerol3-Phosphate(G3P)assay:
4.1 Add50μLofG3Passaymixture(fromStep2.2)toeachwelloftheG3Pstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalG3Passayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLsampleand25μLofG3Passaymixtureperwell.
4.2 Incubatethereactionatroomtemperaturefor30minto1hour,protectedfromlight.
4.3 Monitortheabsorbanceincreasewithanabsorbancemicroplatereaderat575nm(orratioof570nm/610nm).
| References&Citations |  PrinterFriendlyVersion |
1. SantoroA,CappelloAR,MadeoM,MartelloE,IacopettaD,DolceV.(2011)Interactionoffosfomycinwiththeglycerol3-phosphatetransporterofEscherichiacoli.BiochimBiophysActa,1810,1323.
2. GaoY,PanY.(2011)CCAAT/enhancerbindingprotein-betanegativelyregulatestheexpressionofglycerol-3-phosphatedehydrogenase1inpigPK-15cells.JApplGenet,52,451.
3. FrohlichKM,RobertsRA,HousleyNA,AudiaJP.(2010)Rickettsiaprowazekiiusesansn-glycerol-3-phosphatedehydrogenaseandanoveldihydroxyacetonephosphatetransportsystemtosupplytriosephosphateforphospholipidbiosynthesis.JBacteriol,192,4281.
4. ChenH,JiangJG,WuGH.(2009)EffectsofsalinitychangesonthegrowthofDunaliellasalinaanditsisozymeactivitiesofglycerol-3-phosphatedehydrogenase.JAgricFoodChem,57,6178.
5. WydyshEA,MedghalchiSM,VadlamudiA,TownsendCA.(2009)Designandsynthesisofsmallmoleculeglycerol3-phosphateacyltransferaseinhibitors.JMedChem,52,3317.
6. AnejaKK,GuhaP,ShilpiRY,ChakrabortyS,SchrammLM,HaldarD.(2008)Thepresenceofdistalandproximalpromotersforratmitochondrialglycerol-3-phosphateacyltransferase.ArchBiochemBiophys,470,35.
7. RoyA,GuhaN,VerasID,ChakrabortyS,HaldarD.(2003)Stimulationofratlivermitochondrialsn-glycerol-3-phosphateacyltransferasebypolymyxinBviaenhancedextractionoflysophosphatidicacid.Lipids,38,965.
8. BadurinaDS,Zolli-JuranM,BrownED.(2003)CTP:glycerol3-phosphatecytidylyltransferase(TarD)fromStaphylococcusaureuscatalyzesthecytidylyltransferviaanorderedBi-BireactionmechanismwithmicromolarK(m)values.BiochimBiophysActa,1646,196.
9. KannanL,KnudsenJ,JollyCA.(2003)Agingandacyl-CoAbindingproteinaltermitochondrialglycerol-3-phosphateacyltransferaseactivity.BiochimBiophysActa,1631,12.
10. BerradaW,NayaA,IddarA,BourhimN.(2002)Purificationandcharacterizationofcytosolicglycerol-3-phosphatedehydrogenasefromskeletalmuscleofjerboa(Jaculusorientalis).MolCellBiochem,231,117.
