| 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 |
Note:1.Toachievethebestresults,it’sstronglyrecommendedtousetheblackplates.
2.Thawonevialofeachkitcomponentatroomtemperaturebeforestartingtheexperiment.
1.PrepareAmplite™HRPsubstratestocksolution(200X):
Add50µLofDMSOintothevialofAmplite™HRPsubstrate(ComponentA)tomake200Xstocksolution.
Note:Makeasingleusedaliquots,andstoreunused200XAmplite™HRPsubstratestocksolutionat-20ºC,avoidlightandrepeatfreeze-thawcycles.
2.Prepareglycerolassaymixture:
2.1 Add5mLofAssayBuffer(ComponentC)intoabottleofEnzymeMix(ComponentB),mixthemwell.
2.2 Add25µLofAmplite™HRPsubstratestocksolution(fromStep1)intothebottleofComponentB+C(fromStep2.1),andmixthemwelltomakeglycerolassaymixture(ComponentA+B+C).
Note1.Thisglycerolassaymixtureisenoughforone96-wellplate.Itisnotstable,useitpromptly.
Note2.OnecandivideunusedComponentB+Cintosingleusealiquotsandstoredat-20oC.
3.PrepareGlycerolstandardstocksolution:
Add1mLofddH2Oor1×PBSbufferintothevialofglycerolstandard(ComponentD)tomake1mg/mLglycerolstandardstocksolution.
Note:Theunusedglycerolstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
4.Prepareserialdilutionsofglycerolstandard(0to10µg/mL):
4.1 Add10μLofglycerolstandardstocksolution(1mg/mL,fromStep3)into990µL1×PBSbuffertogenerate10µg/mLstandardsolution.
4.2 Andthenperform1:2serialdilutionstogetapproximately5,2.5,1.25,0.625,0.313,0.156and0µg/mLseriallydilutedglycerolstandards.
4.3 Addserialdilutionsofglycerolstandardandglycerolcontainingtestsamplesintoaclearbottom96-wellmicroplateasdescribedinTables1and2.
Table1Layoutofglycerolstandardsandtestsamplesinaclearbottom96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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G1 | G1 | …. | …. | …. | …. |
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G2 | G2 |
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G3 | G3 |
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G4 | G4 |
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G5 | G5 |
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G6 | G6 |
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G7 | G7 |
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Note:G=GlycerolStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
GlycerolStandard | BlankControl | TestSample |
SerialDilutions*:50μL | 1×PBSBuffer:50μL | 50μL |
*Note:Addtheseriallydilutedglycerolstandardsfromapproximately0.156µg/mLto10µg/mLintowellsfromG1toG7induplicate.
5.Runglycerolassay:
5.1 Add50μLofglycerolassaymixture(fromStep2.2)toeachwellofglycerolstandard,blankcontrol,andtestsamples(seeStep4.3)tomakethetotalvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofglycerolassaymixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor30minutesto1hour,protectedfromlight.
5.3 MonitortheabsorbanceincreasewithanabsorbanceplatereaderwithpathcheckatODof575nm.
| References&Citations |  PrinterFriendlyVersion |
1. WuQ,LiJZ,ZhaoTY,LiTB,WangJX,SuiY.(2014)Epigallocatechin-3-gallate(EGCG)inhibits3-hydroxy-3-methylglutaryl-CoAreductaseinthepresenceofglycerol.PakJPharmSci,27,1905.
2. WilliasSP,ChauhanS,MotinVL.(2014)FunctionalcharacterizationofYersiniapestisaerobicglycerolmetabolism.MicrobPathog,76C,33.
3. LoweHI,ToyangNJ,WatsonCT,BryantJ.(2014)Synthesisofsubstituted1,3-diestersofglycerolusingwittigchemistry.NatProdCommun,9,687.
4. RoheA,GollnerC,ErdmannF,SipplW,SchmidtM.(2014)Theglycoglycerolipid1,2-dipalmitoyl-3-(N-palmitoyl-6"-amino-6"-deoxy-alpha-d-glucosyl)-sn-glycerolisnoinhibitorofthehumanMyt1kinase.JEnzymeInhibMedChem,1.
5. LinkeD,LehnertN,NimtzM,BergerRG.(2014)AnalcoholoxidaseofPhanerochaetechrysosporiumwithadistinctglyceroloxidaseactivity.EnzymeMicrobTechnol,61-62,7.
6. DongY,MaY,YanK,ShenL,WangX,XuY,HeG,WuY,LuJ,YangZ,FengF.(2014)Quantitativeanalysisofglycerollevelsinhumanurinebyliquidchromatography-tandemmassspectrometry.JChromatogrBAnalytTechnolBiomedLifeSci,957,30.
7. AbadS,PerezX,PlanasA,TuronX.(2014)Rapidmonitoringofglycerolinfermentationgrowthmedia:Facilitatingcrudeglycerolbioprocessdevelopment.Talanta,121,210.
8. JeongCY,HanYD,YoonJH,YoonHC.(2014)Bioelectrocatalyticsensorfortriglyceridesinhumanskinsebumbasedonenzymaticcascadereactionoflipase,glycerolkinaseandglycerophosphateoxidase.JBiotechnol,175,7.
9. LaConteLE,ChavanV,MukherjeeK.(2014)Identificationandglycerol-inducedcorrectionofmisfoldingmutationsintheX-linkedmentalretardationgeneCASK.PLoSOne,9,e88276.
10. YunY,LiuZ,ZhangJ,ShimWB,ChenY,MaZ.(2014)TheMAPKKFgMkk1ofFusariumgraminearumregulatesvegetativedifferentiation,multiplestressresponse,andvirulenceviathecellwallintegrityandhigh-osmolarityglycerolsignalingpathways.EnvironMicrobiol,16,2023.
