Overview

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Ex/Em(nm)	646/659
MW	422.20
CAS#	N/A
Solvent	Water
Storage	F/D/L
Category	EnzymeDetection Phosphatases
Related	ProteinPhosphatase BiochemicalAssays

Phosphatase-catalyzedhydrolysisofSunRedphosphate(SRP)yieldstheSunRedfluorophorethatcanbeexcitedwiththe633nmlaserwithemissionof~660nm.AlthoughSunRedisreADIlyexcitedat633nmwithredemissionof~660nm,SRPhasveryminimalabsorptionat633nmwithoutredemission,makingSRPoneofthemostsensitiveNIRphosphatasesensors.PleasedonotuseDMSOtomakestocksolutionsinceitsignificantlyincreasesassaybackground.

Spectrum

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareSunRed™Phosphateworkingsolution:

1.1   Preparea2to10mMstocksolutionofSunRed™Phosphateinsterilewater.Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandfrozenat<-20^oC.

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

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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.

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