| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 646/659 |
| MW | 422.20 |
| CAS# | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category | EnzymeDetection Phosphatases |
| Related | ProteinPhosphatase BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareSunRed™Phosphateworkingsolution:
1.1 Preparea2to10mMstocksolutionofSunRed™Phosphateinsterilewater.Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandfrozenat<-20oC.
Note1:DonotuseDMSO,ETOHorMETHtomakestocksolutionsinceitsignificantlyincreasesassaybackground.
Note2:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
1.2 Preparea2XSunRed™Phosphateworkingsolution: Onthedayoftheexperiment,eitherdissolveSunRed™PhosphatesolidinsterileH2OorthawanaliquotoftheSunRed™Phosphatestocksolutionatroomtemperature.Preparea2Xworkingsolutionof10to50µMin100mMTrisbufferorbufferofyourchoice,pH8to9.SunRed™Phosphatefinalconcentrationof5to25µMisrecommendedformeasuringalkalinephosphataseactivityinsolution.
2.Runalkalinephosphataseassayinsupernatants:
2.1 Add50µLof2XSunRed™Phosphateworkingsolution(fromStep1.2)intoeachwellofthealkalinephosphatasestandard,blankcontrol,andtestsamplestomakethetotalalkalinephosphataseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25µLofsampleand25µLof2XSunRed™Phosphateworkingsolutionintoeachwell.
2.2 Incubatethereactionfor30to120minutesatthedesiredtemperature,protectedfromlight.
2.3 MonitorthefluorescenceincreaseatEx/Em=620/660nm(cutoffat640nm)withafluorescenceplatereader.
2.4 Thefluorescenceinblankwells(withtheassaybufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosewellswiththeH2O2reactions.
| References&Citations |  PrinterFriendlyVersion |
1. MarchettiS,OnoriG,CamettiC.(2005)DNAcondensationinducedbycationicsurfactant:aviscosimetryanddynamiclightscatteringstudy.JPhysChemB,109,3676.
2. MartinK,HartC,LiuJ,LeungWY,PattonWF.(2003)SimultaneoustrichromaticfluorescencedetectionofproteinsonWesternblotsusinganamine-reactivedyeincombinationwithalkalinephosphatase-andhorseradishperoxidase-antibodyconjugates.Proteomics,3,1215.
3. TopKP,HatlebergG,BerggrenKN,RyanD,KemperC,HauglandRP,PattonWF.(2001)Green/reddualfluorescencedetectionoftotalproteinandalkalinephosphate-conjugatedprobesonblottingmembranes.Electrophoresis,22,896.
4. LeiraF,VieitesJM,VieytesMR,BotanaLM.(2000)Characterizationof9H-(1,3-dichlor-9,9-dimethylacridin-2-ona-7-yl)-phosphate(DDAO)assubstrateofPP-2Ainafluorimetricmicroplateassayfordiarrheticshellfishtoxins(DSP).Toxicon,38,1833.
5. Vazquez-ContrerasE,deGomez-PuyouMT,DreyfusG.(1996)AcolumncentrifugationmethodforthereconstitutioninliposomesofthemitochondrialF0F1ATPsynthase/ATPase.ProteinExprPurif,7,55.
6. ValerioM,DiolezP,HarauxF.(1993)TheelectRochemical-proton-gradient-activatedstatesofF0F1ATPaseinplantmitochondriaasrevealedbydetergents.EurJBiochem,216,565.
