eEnzyme/NAD/NADH Ratio Assay Kit (Red Fluorescence)/CAN226/1 Ea

CA-N226
Name	Elite^TMNAD/NADHRatioAssayKit(RedFluorescence)
Description	TheElite^TMFluorimetricNAD/NADHRatioAssayKitprovidesaconvenientmethodforsensitivedetectionofNAD,NADHandtheirratio;thereisnoneedtopurifyNAD/NADHfromsamplemix.Ithasverylowbackgroundsinceitisrunintheredvisiblerangethatsignificantlyreducestheinterferenceresultedfrombiologicalsamples.
Application	Theassaycanbeconvenientlyperformedina96-wellor384-wellmicrotiter-plateformat.ItssignalcanbeeasilyreadbyeitherafluorescencemicroplatereaderatEx/Em=530-570/590-600nm(maximumEx/Em=540/590nm)oranabsorbancemicroplatereaderat~576nm.ThiskitprovidesNADandNADHextractionbuffer,andcelllysisbufferforyourconvenience.IthasbeenfrequentlyusedfordeterminingNAD/NADHfromcelllysates.
Size	250assaysin96-wellplates
Ex/Em	540/590nm
Detection	Fluorescencemicroplatereader

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FrequentlyAskedQuestions

1.CanweusethisratioassaykittoalsodetectNADorNADHseparately?

Answer:Yes.YoucouldmeasuretheNADHamountwithComponentD(NADHExtractionSolution),andmeasuretheNADamountwithNADExtractionSolution.Butaccordingtoourexperience,theNADconcentrationsincellsarealothigherthantheNADHconcentrations.WesuggesttomeasuretheTotalNAD+NADHamountandtheNADamount,thentocalculatetheNADHamountbysubtraction,whichgivesmoreaccurateresults.

2.WhyPBSisusedtodilutetheStandard?Sincethesamplesareinthelysisbuffer,doyouthinkIshouldusethelysisbuffertodilutethem?

Answer:PBSiscommonlyused.Peopleusuallyusethelysisbuffertolysethecells,thenusePBStodilutethesamples.ThepointisthatyoushouldkeepthesameconcentrationofthelysisbufferforthesamplesandtheStandards.

3.Ididacomparison,anditshowedthatthePBSbasedStandardcurvelookedbetterthanthelysisbufferbasedStandardcurve.Whatdoyouthinkofthis?

Answer:Thebubblesinthelysisbuffermaycausetheinconsistencyofthereadings.

4.WhenIdoduplicatesofStandards,thereadingvariesalotinthelowrange(0.2uMorlower).Whydoesthathappen?

Answer:Itisnormal.Therearemanyfactorsthatwouldcauseinconsistentreadings,i.e.bubblesinthewells,thevolumes....Sotrydoingmorereplicates.

5.Sometimesforthe2setsofStandardsIsetup,theylookgood(R2=0.99)individuallybutthecurveismessedup(R2=0.95)whenmerged.SowhichcurveshouldIusetoquantifymydata?

Answer:Youllneedtousethecurvesetuponthesameplatewithyoursamples.

6.Wedonthavefluorescencemicroplatereader,socanweuseabsorbancemicroplatereader?

Answer:Yes.Buttheabsorbancereadingshavemuchlowersensitivitycomparedtothefluorescencereadings.Tryusingtheratioof570to610toincreasethesensitivity.

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eEnzyme/NAD/NADH Ratio Assay Kit (Red Fluorescence)/CAN226/1 Ea