Overview | ![]() PrinterFriendlyVersion |
Ex/Em(nm) | 360/450 |
MW | 352.55 |
CAS# | N/A |
Solvent | Water |
Storage | F/D/L |
Category | EnzymeDetection Phosphatases |
Related | ProteinPhosphatase |
Spectrum | AdvancedSpectrumViewer |
1.Prepareworkingsolution:
1.1 Preparea2to10mMstocksolutioninanappreciatesolvent(seeTable).Thestocksolutionshouldbeusedpromptly.Anyunusedsolutionneedtobealiquotedandfrozenat<-20oC.
Note1:DonotuseDMSO,ETOHorMETHtomakestocksolutionofSunRed™Phosphate(cat#11629)sinceitsignificantlyincreasesassaybackground.
Note2:Avoidrepeatedfreeze-thawcycles,andprotectfromlight.
1.2 Preparea2XPhosphateworkingsolution: Onthedayoftheexperiment,eitherdissolvethesubstrateinanappreciatesolventorthawanaliquotofthestocksolution(fromStep1.1)atroomtemperature.Preparea2Xworkingsolutionof10to50µMin100mMTrisbufferorbufferofyourchoice,pH8to9(notphosphatebuffer).
2.Runphosphataseassayinsupernatants:
2.1 Add50µLof2XPhosphateworkingsolution(fromStep1.2)intoeachwellofthephosphatasestandard,blankcontrol,andtestsamplestomakethetotalphosphataseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25µLofsampleand25µLof2XPhosphateworkingsolutionintoeachwell.
2.2 Incubatethereactionfor30to120minutesatthedesiredtemperature,protectedfromlight.
2.3 Monitorthefluorescenceincreaseatanappropriatefiltersets(Seetable)withafluorescenceplatereader.
2.4 Thefluorescenceinblankwells(withtheassaybufferonly)isusedasacontrol,andissubtractedfromthevaluesforthosewellswiththephosphatasereactions.
References&Citations | ![]() PrinterFriendlyVersion |
1. BednarskaK,KlinkM,SulowskaZ.(2006)Applicationofintracellularalkalinephosphataseactivitymeasurementindetectionofneutrophiladherenceinvitro.MediatorsInflamm,2006,19307.
2. WohlsenT,BaylissJ,GrayB,BatesJ,KatouliM.(2006)EvaluationofanalternativemethodfortheenumerationandconfirmationofClostridiumperfringensfromtreatedanduntreatedsewages.LettApplMicrobiol,42,438.
3. AndersonBH,MagdoffFR.(2005)Relativemovementandsoilfixationofsolubleorganicandinorganicphosphorus.JEnvironQual,34,2228.
4. RinconV,CorredorA,Martinez-GutierrezM,CastellanosJE.(2005)Fluorometriccell-ELISAforquantifyingrABIesinfectionandheparininhibition.JVirolMethods,127,33.
5. HoehamerCF,MazurCS,WolfeNL.(2005)PurificationandpartialcharacterizationofanacidphosphatasefromSpirodelaoligorrhizaanditsaffinityforselectedorganophosphatepesticides.JAgricFoodChem,53,90.
6. ShroyerML,BhuniaAK.(2003)Developmentofarapid1-hfluorescence-basedcytotoxicityassayforListeriaspecies.JMicrobiolMethods,55,35.
7. ChruszczM,LaidlerP,MonkiewiczM,OrtlundE,LebiodaL,LewinskiK.(2003)CrystalstructureofacovalentintermediateofendogenoushumanarylsulfataseA.JInorgBiochem,96,386.
8. BautersTG,PelemanR,DhontM,VanhaesebrouckP,NelisHJ.(2003)EnzymaticdifferentiationofCandidaparapsilosisfromotherCandidaspp.inamembranefiltrationtest.JMicrobiolMethods,53,11.
9. BouaichaN,MaatoukI,VincentG,LeviY.(2002)Acolorimetricandfluorometricmicroplateassayforthedetectionofmicrocystin-LRindrinkingwaterwithoutpreconcentration.FoodChemToxicol,40,1677.
10. BarkhudaryanN,GambarovS,GyulbayazyanT,NahapetyanK.(2002)LVV-hemorphin-4modulatesCa2+/calmodulin-dependentpathwaysintheimmunesystembythesamemechanismasinthebrain.JMolNeurosci,18,203.
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