Overview | PrinterFriendlyVersion |
Ex/Em(nm) | None/None |
MW | N/A |
CAS# | N/A |
Solvent | N/A |
Storage | F/D/L |
Category | MicroBIOLOGy iFluorRapidTests |
Related | BiochemicalAssays |
1) Make1mMstocksolutionsoftheselectedstandardandtestsamplesusingpropersolvent(suchaswaterorDMSO).
2) Recordtheabsorbancespectraoftheselectedstandardandtestsamples(0.1to5µM)usingthedilutedsolutionspreparedfromtheirstocksolutions(fromStep#1)withanaqueousbuffer(suchasPBSorTRIS).
3) Selecttheexcitationwavelengthbasedontheabsorptionmaxima(fromStep#2).Werecommendtousethisfollowingformulatoselectthedesiredexcitationwavelength:
Desiredexcitation(nm)=theshorterabsorptionmaximum*–50nm
(*Selectedfromeitherthereferencestandardortestsample.Forexample,ifyourtestsamplehasabsorptionmaximumat500nm,andtheselectedreferencestandardhastheabsorptionmaximumat490nm,youshouldselect440nm(490nm-50nm)asexcitationwavelength)
4) Adjusttheconcentrationsoftheselectedreferencestandardandtestsamplestohavethesameabsorbance(between0.2to0.8)attheselectedexcitationwavelength.
Note:Itmightbeeasiertochoosetheisosbesticpointofthereferencestandardandtestsampleabsorptionspectra.
5) Diluteboththeselectedreferencestandardandtestsampleby10times(mustbedilutedbythesameproportion)usingthesamebufferorpurewater.
6) Recordthefluorescencespectraofthesamedilutedsolutions(fromStep#5)inthe10mmfluorescencecuvetteusingafluorescencespectrophotometeroramicroplatereaderthatcangeneratecorrectedfluorescencespectrum.
Note:thatallthefluorescencespectramustberecordedwiththesameinstrumentsettings.Changinginstrumentsettingsbetweensampleswillinvalidatethequantumyieldmeasurement.
7) Calculatetheintegratedfluorescenceintensity(theareaofthefluorescencespectrum)fromthefullycorrectedfluorescencespectrum(seeyourinstrumentinstruction).
8) Calculatethefluorescencequantumyieldusingthefollowingequation:
Φx=Φs
Φxand Φs arethefluorescencequantumyieldsoftheselectedreferencestandardandtestsamplerespectively.AxandAsaretheintegratedfluorescenceintensity(thecurvearea)ofselectedreferencestandardandtestsamplerespectively.
References&Citations | PrinterFriendlyVersion |
1. GrabolleM,ZieglerJ,MerkulovA,NannT,Resch-GengerU.(2008)StabilityandfluorescencequantumyieldofCdSe-ZnSquantumdots--influenceofthethicknessoftheZnSshell.AnnNYAcadSci,1130,235.
2. BahadduriPM,RayA,KhandelwalA,SwaanPW.(2008)Designofhigh-affinitypeptideconjugateswithoptimizedfluorescencequantumyieldasMarkersforsmallpeptidetransporterPEPT1(SLC15A1).BioorgMedChemLett,18,2555.
3. WeiYJ,LiN,QinSJ.(2004)Fluorescencespectraandfluorescencequantumyieldofsulfosalicylicacid.GuangPuXueYuGuangPuFenXi,24,647.
4. DiamondKR,FarrellTJ,PattersonMS.(2003)Measurementoffluorophoreconcentrationsandfluorescencequantumyieldintissue-simulatingphantomsusingthreediffusionmodelsofsteady-statespatiallyresolvedfluorescence.PhysMedBiol,48,4135.
5. BlaisJ,DoukiT,VignyP,CadetJ.(1994)Fluorescencequantumyielddeterminationofpyrimidine(6-4)pyrimidonephotoadducts.PhotochemPhotobiol,59,402.
6. KvamE,MoanJ.(1990)Acomparisonofthreephotosensitizerswithrespecttoefficiencyofcellinactivation,fluorescencequantumyieldandDNAstrandbreaks.PhotochemPhotobiol,52,769.
7. TsinAT,Pedrozo-FernandezHA,GallasJM,ChambersJP.(1988)ThefluorescencequantumyieldofvitaminA2.LifeSci,43,1379.
8. KubotaY,SteinerRF.(1977)FluorescencedecayandquantumyieldcharacteristicsofacridineorangeandproflavineboundtoDNA.BiophysChem,6,279.
9. BorisovaOF,PotapovAP,SurovayaAN,TrubitsinSN,Vol"kenshteinMV.(1974)DependenceofthefluorescencequantumyieldofcomplexesofacriflavinewithtRNAonitsstructure.MolBiol,7,411.
10. BorgKO,LagerstromPO.(1970)Quantumyieldoffluorescenceofdimethylprotriptyliniumbromide.ActaPharmSuec,7,567.
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