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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/FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*/11600/5 mg
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AAT Bioquest/FDP [Fluorescein diphosphate, tetraammonium salt] *CAS 217305-49-2*/11600/5 mg
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Overview
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Ex/Em(nm)490/514
MW560.39
CAS#217305-49-2
SolventWater
StorageF/D/L
CategoryEnzymeDetection
Phosphatases
RelatedProteinPhosphatase
BiochemicalAssays
Uponinteractionwithphosphatasesthecolorlessandnon-fluorescentFDPishydrolyzedtohighlyfluorescentfluorescein,whichexhibitsexcellentspectralpropertiesthatmatchtheoptimaldetectionwindowofmostfluorescenceinstrumentsthatareequippedwiththeArgonlaserexcitation.Alternatively,FDPcanalsobeusedtodetectphosphatasesinachromogenicmodesincetheenzymaticproduct(fluorescein)exhibitsalargeextinctioncoefficient(closeto100,000cm-1mol-1).Insomeliterature,FDPwasconsideredtobeoneofthemostsensitivefluorogenicphosphatasesubstrates.FDPhasbeenwidelyusedinvariousELISAassays.Additionallyitisalsousedtodetecttyrosinephosphatases.FDPisthermallyunstable,andspecialcautionsneedbeexcisedforstoringthesolidsampleandstocksolutions.
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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.   HillRT,ShearJB.(2006)Enzyme-nanoparticlefunctionalizationofthree-dimensionalproteinscaffolds.AnalChem,78,7022.

2.   KarlssonA,SottK,MarkstromM,DavidsonM,KonkoliZ,OrwarO.(2005)Controlledinitiationofenzymaticreactionsinmicrometer-sizedbiomimeticcompartments.JPhysChemBCondensMatterMaterSurfInterfacesBiophys,109,1609.

3.   WardDG,TaylorM,LilleyKS,CavieresJD.(2006)TNP-8N3-ADPphotoaffinitylabelingoftwoNa,K-ATPasesequencesunderseparateNa+plusK+control.Biochemistry,45,3460.

4.   DevaderC,WebbRJ,ThomasGM,DaleL.(2006)Xenopusapyrase(xapy),asecretednucleotidasethatisexpressedduringearlydevelopment.Gene,367,135.

5.   FarrellS,HalsallHB,HeinemanWR.(2005)ImmunoassayforB.globigiisporesasamodelfordetectingB.anthracissporesinfinishedwater.Analyst,130,489.

6.   MontalibetJ,SkoreyKI,KennedyBP.(2005)Proteintyrosinephosphatase:enzymaticassays.Methods,35,2.

7.   HassepassI,HoffmannI.(2004)AssayingCdc25phosphataseactivity.MethodsMolBiol,281,153.

8.   BramkampM,GasselM,AltendorfK.(2004)FITCbindingsiteandp-nitrophenylphosphataseactivityoftheKdp-ATPaseofEscherichiacoli.Biochemistry,43,4559.

9.   BirisN,AbatzisM,MitsiosJV,Sakarellos-DaitsiotisM,SakarellosC,TsoukatosD,TselepisAD,MichalisL,SiderisD,KonidouG,SoteriadouK,TsikarisV.(2003)Mappingthebindingdomainsofthealpha(IIb)subunit.Astudyperformedontheactivatedformoftheplateletintegrinalpha(IIb)beta(3).EurJBiochem,270,3760.

10.   RiderDA,YoungSP.(2003)MeasuringthespecificactivityoftheCD45proteintyrosinephosphatase.JImmunolMethods,277,127.


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