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AAT Bioquest/Amplite™ Fluorimetric Beta-Hydroxybutyrate (Ketone Body) Assay Kit/13831/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy CellMetabolism |
| Related | RedoxEnzymes BiochemicalAssays |
1.PrepareNADstocksolution(100×):
Add100µLofH2OintothevialofNAD(ComponentC)tomake100×NADstocksolutions.
2.Prepareβ-HBstandardstocksolution:
Add1mLofH2Oor1×PBSbufferintothevialofβ-HBstandard(ComponentD)tomake100mMβ-HBstandardstocksolution.
Note:Theunusedβ-HBstandardstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
3.Prepareserialdilutionsofβ-HBstandard:
3.1 Add10μLofβ-HBstandardstocksolution(100mM,fromStep2)into990µL1×PBSbuffertogenerate1mMβ-HBstandardsolution.
Note:Dilutedβ-HBstandardsolutionisunstable,andshouldbeusedwithin4hours.
3.2 Take200μLof1mMβ-HBstandardsolutiontoperform1:3serialdilutionsinPBStogetapproximately300,100,30,10,3,1and0μMserialdilutionsofβ-HBstandard.
3.3 Addserialdilutionsofβ-HBstandardandβ-HBcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Table1Layoutofβ-HBstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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HB1 | HB1 | …. | …. | …. | …. |
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HB2 | HB2 |
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HB3 | HB3 |
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HB4 | HB4 |
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HB5 | HB5 |
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HB6 | HB6 |
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HB7 | HB7 |
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Note:HB=β-HBStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
β-HBStandard | BlankControl | TestSample |
SerialDilutions*:50μL | 1×PBSBuffer:50μL | 50μL |
*Note:Addtheseriallydilutedβ-HBstandardsfromapproximately1μMto1mMintowellsfromHB1toHB7induplicate.
4.Prepareβ-HBassaymixture:
4.1 Add5mLofAssayBuffer(ComponentB)intoonebottleofEnzymeMix(ComponentA).
4.2 Add50µLNADstocksolution(ComponentC,fromStep1)intothebottleofComponentA+B(fromStep4.1),andmixwelltomakeβ-HBassaymixture(ComponentA+B+C).
Note:Thisβ-HBassaymixtureisenoughforone96-wellplate.Theunusedβ-HBassaymixtureshouldbedividedinto
singleusealiquotsandstoredat-20oC.
5.Runβ-HBassay:
5.1 Add50μLofβ-HBassaymixture(fromStep4.2)toeachwellofβ-HBstandard,blankcontrol,andtestsamples(seeStep3.3)tomakethetotalvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofβ-HBassaymixtureintoeachwell.
5.2 Incubatethereactionatroomtemperaturefor10-30minutes,protectedfromlight.
5.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=530-570/590-600nm(optimalEx/Em=540/590nm,cutoffat570nm).
| References&Citations |  PrinterFriendlyVersion |
1. Zharkova,II,StaroverovaOV,VoinovaVV,AndreevaNV,ShushckevichAM,SklyanchukED,KuzmichevaGM,BespalovaAE,AkulinaEA,ShaitanKV,OlkhovAA.(2014)[Biocompatibilityofelectrospunpoly(3-hydroxybutyrate)anditscompositesscaffoldsfortissueengineering.].BiomedKhim,60,553.
2. WangW,LanP.(2014)Surfaceglycosylationofpoly(3-hydroxybutyrate-co-4-hydroxybutyrate)membraneforselectiveadsorptionoflow-densitylipoprotein.JBiomaterSciPolymEd,25,2094.
3. JohanssonJ,GronbladhA,HallbergM.(2014)Gamma-hydroxybutyrate(GHB)inducescognitivedeficitsandaffectsGABABreceptorsandIGF-1receptorsinmalerats.BehavBrainRes,269,164.
4. WeiT,TianW,LiuF,XieG.(2014)Protectiveeffectsofexogenousbeta-hydroxybutyrateonparaquattoxicityinratkidney.BiochemBiophysResCommun,447,666.
5. VogensenSB,MarekA,BayT,WellendorphP,KehlerJ,BundgaardC,FrolundB,PedersenMH,ClausenRP.(2013)Newsynthesisandtritiumlabelingofaselectiveligandforstudyinghigh-affinitygamma-hydroxybutyrate(GHB)bindingsites.JMedChem,56,8201.
6. ZhengJ,LiD,YuanL,LiuX,ChenH.(2013)Lotus-leaf-liketopographypredominatesoveradsorbedECMproteinsinpoly(3-hydroxybutyrate-co-3-hydroxyhexanoate)surface/cellinteractions.ACSApplMaterInterfaces,5,5882.
7. BonartsevAP,YakovlevSG,Zharkova,II,BoskhomdzhievAP,BagrovDV,MyshkinaVL,MakhinaTK,KharitonovaEP,SamsonovaOV,FeofanovAV,VoinovaVV,ZernovAL,EfremovYM,BonartsevaGA,ShaitanKV,KirpichnikovMP.(2013)Cellattachmentonpoly(3-hydroxybutyrate)-poly(ethyleneglycol)copolymerproducedbyAzotobacterchroococcum7B.BMCBiochem,14,12.
8. LimJ,ChongMS,TeoEY,ChenGQ,ChanJK,TeohSH.(2013)Biocompatibilitystudiesandcharacterizationofpoly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/polycaprolactoneblends.JBiomedMaterResBApplBiomater,101,752.
9. LomasAJ,WebbWR,HanJ,ChenGQ,SunX,ZhangZ,ElHajAJ,ForsythNR.(2013)Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)/collagenhybridscaffoldsfortissueengineeringapplications.TissueEngPartCMethods,19,577.
10. ChengB,LuH,BaiB,ChenJ.(2013)d-beta-HydroxybutyrateinhibitedtheapoptosisofPC12cellsinducedbyH2O2viainhibitingoxidativestress.NeuRochemInt,62,620.
