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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/MUP [4-Methylumbelliferyl phosphate, free acid] *CAS 3368-04-5*/11614/25 mg
产品编号:11614
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AAT Bioquest/MUP [4-Methylumbelliferyl phosphate, free acid] *CAS 3368-04-5*/11614/25 mg
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Ex/Em(nm)360/449
MW256.15
CAS#3368-04-5
SolventWater
StorageF/D/L
CategoryEnzymeDetection
Phosphatases
RelatedProteinPhosphatase
BiochemicalAssays
Afluorogenicsubstrateforphosphatases.Itiswidelyusedfordetectingphosphatasesinsolution.HoweverthisphosphatasesubstrateisnotwellsuitedforlivingcellorcontinuousassayssinceMU(4-methylumbelliferone),theenzymaticproduct,whichonlydevelopsmaximumfluorescenceatpHvalueof>10.ThusitisalsodifficulttouseMUPforthedetectionofphosphatasesthathaveacidicoptimalpHrangesuchasacidphosphatases.AATBioquestispleasedtoofferMUPPlusthatisdevelopedtoaddressthispHlimitationassociatedwithMUPsubstrates.
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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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
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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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