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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/pNPP [4-Nitrophenyl phosphate, disodium salt] *CAS 4264-83-9*/11619/25 mg
产品编号:11619
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AAT Bioquest/pNPP [4-Nitrophenyl phosphate, disodium salt] *CAS 4264-83-9*/11619/25 mg
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Ex/Em(nm)399/None
MW263.05
CAS#4264-83-9
SolventWater
StorageF/D/L
CategoryEnzymeDetection
Phosphatases
RelatedProteinPhosphatase
BiochemicalAssays
para-Nitrophenylphosphate(pNPP)isachromogenicsubstrateforacidandalkalinephosphataseinELISAassays.InthepresenceofphosphatasespNPPishydrolyzedtogeneratetheyellowpara-nitrophenolthatcanbemeasuredat405nmwithanabsorptionmicroplatereaderoraspectrophotometer.

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.   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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