Overview | ![]() PrinterFriendlyVersion |
Ex/Em(nm) | 399/None |
MW | 263.05 |
CAS# | 4264-83-9 |
Solvent | Water |
Storage | F/D/L |
Category | EnzymeDetection Phosphatases |
Related | ProteinPhosphatase BiochemicalAssays |
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. AliyuIS,IsahHS,AfonjaOA.(2006)ReferenceIntervalofSerumHeat-stableAlkalinePhosphataseActivityinPregnantWomeninZaria.NigerPostgradMedJ,13,31.
2. ChaoTY,LeeSH,ChenMM,NeustadtDH,ChaudhryUA,YamLT,JanckilaAJ.(2005)Developmentofimmunoassaysforserumtartrate-resistantacidphosphataseisoform5a.ClinChimActa,359,132.
3. JanckilaAJ,SimonsRM,YamLT.(2004)Alternativeimmunoassayfortartrate-resistantacidphosphataseisoform5busingthefluorogenicsubstratenaphtholASBI-phosphateandheparin.ClinChimActa,347,157.
4. GuimaraesLH,TerenziHF,JorgeJA,LeoneFA,PolizeliML.(2003)ExtracellularalkalinephosphatasefromthefilamentousfungusAspergilluscaespitosus:purificationandbiochemicalcharacterization.FoliaMicrobiol(Praha),48,627.
5. CroninA,MowbrayS,DurkH,HomburgS,FlemingI,FisslthalerB,OeschF,ArandM.(2003)TheN-terminaldomainofmammaliansolubleepoxidehydrolaseisaphosphatase.ProcNatlAcadSciUSA,100,1552.
6. KokadoA,ArakawaH,MaedaM.(2002)ChemiluminescentassayofalkalinephosphataseusingdihydroxyacetonephosphateassubstratedetectedwithLucigenin.Luminescence,17,5.
7. LauCK,LoSC,LiW,ChurchichDR,KwokF,ChurchichJE.(1998)Partiallyfoldedconformationsofinositolmonophosphataseendowedwithcatalyticactivity.JProteinChem,17,789.
8. RushJS,WaechterCJ.(1998)TopologicalstudiesontheenzymescatalyzingthebiosynthesisofGlc-P-dolicholandthetriglucosylcapofGlc3Man9GlcNAc2-P-P-dolicholinmicrosomalvesiclesfrompigbrain:useoftheprocessingglucosidasesI/IIaslatencyMarkers.GlycoBIOLOGy,8,1207.
9. HuangTM,HungHC,ChangTC,ChangGG.(1998)Solventkineticisotopeeffectsofhumanplacentalalkalinephosphataseinreversemicelles.BiochemJ,330(Pt1),267.
10. TurckF,KozmaSC,ThomasG,NagyF.(1998)Aheat-sensitiveArABIdopsisthalianakinasesubstitutesforhumanp70s6kfunctioninvivo.MolCellBiol,18,2038.
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