| 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 |
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