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Ex/Em(nm)	360/449
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	EnzymeDetection Phosphatases
Related	BiochemicalAssays

AlkalinephosphataseisahighlysensitiveenzymeforELISA,immuno-histochemical,Northern,SouthernandWesternblotapplications.ItiswidelyusedinvariousBIOLOGicalassays(inparticular,immunoassays)andELISA-baseddiagnostics.ThisAmplite™AlkalinePhosphataseAssayKitusesMUP,afluorogenicphosphatasesubstrate,toquantifyalkalinephosphataseactivityinsolutions,incellextractsaswellasonsolidsurfaces(suchasPVDFmembranes).Thekitprovidesalltheessentialcomponentswithouroptimized"mixandread"assayprotocolthatiscompatIBLewithHTSliquidhandlinginstruments.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Prepare250XMUPPlus™stocksolutions:

Add100µLofsterileH₂OintothevialofMUPPLus™(ComponentA).TheMUPPLus™stocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandrefrozenat-20^oC.

Note:Avoidrepeatedfreeze-thawcycles.

2.Prepareassayreactionmixture:

Prepareassayreactionmixtureaccordingtothefollowingtableandkeepfromlight.

Table1Assayreactionmixtureforone96-wellplate

Components	Volume
MUPPlus™(250X,fromStep1.1)	20μL
AssayBuffer(ComponentB)	5mL
Totalvolume	5mL

Note:Preparefreshreactionmixtureforeachexperiment.

 

3.Prepareseriallydilutedalkalinephosphatasestandards(0to100mU/mL):

3.1   Add100μLofdistilledH₂Owith0.1%BSA(H₂O-0.1%BSA)toalkalinephosphatasestandard(ComponentC,10units)togeneratea100units/mLalkalinephosphatasestandardsolution.

Note:Thealkalinephosphatasestandardsolutionisnotstable.Aliquotandstoreunusedsolutionat‑ 20 ^oC.Avoidrepeatedfreeze-thawcycles.

 

3.2   Add10μLof100units/mLalkalinephosphatasestandardsolution(fromStep3.1)into990µLofH₂O-0.1%BSAtogeneratea1,000mU/mLalkalinephosphatasestandardsolution.

 

3.3   Take100μLof1,000mU/mLsolution(fromStep3.2)toperform1:10andthen1:3serialdilutionstoget100,30,10,3,1,0.3,0.1,and0mU/mLserialdilutionsofalkalinephosphatasestandard.

 

3.4   Addseriallydilutedalkalinephosphatasestandardsand/oralkalinephosphatasecontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables2and3.

Note1:Preparecellsortissuesamplesasdesired.

Note2:Unusedportionofdilutedalkalinephosphatasestandardsolutionshouldbediscarded.

 

Table2Layoutofalkalinephosphatasestandardsandtestsamplesinasolidblack96-wellmicroplate

Note:AS=AlkalinePhosphataseStandards;BL=BlankControl;TS=TestSamples.

 

Table3Reagentcompositionforeachwell

*Note:Addtheseriallydilutedalkalinephosphatasestandardsfrom10to0.01mU/mLintowellsfromAS1toAS7induplicate.

 

4.Runalkalinephosphataseassayinsupernatants:

4.1   Add50μLofassayreactionmixture(fromStep2)intoeachwellofalkalinephosphatasestandard,blankcontrol,andtestsamples(seeStep3.4,Table3)togetthetotalalkalinephosphataseassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.

 

4.2   Incubatethereactionatthedesiredtemperaturefor10to30minutes,protectedfromlight.

 

4.3   MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=360±10/450±10nm.

 

5.Runalkalinephosphataseassayincells:

5.1   Treatthecellsasdesired.

5.2   Addequalvolumeofassayreactionmixture(fromStep2)intoeachcellwell(suchas100μL/96-wellplate,or50μL/384-wellplate).

Note:Alternatively,removethegrowthmediumfromthecellplate,andmake1:1dilutionofthe5mLassayreactionmixture(fromStep2,Table1)with5mLdistilledH₂O.ThenAdd100μL(fora96-wellplate)or50uL(fora384-wellplate)of1:1dilutedassayreactionmixtureintothecellwells(fromStep5.2).

5.3   Incubatethereactionatthedesiredtemperaturefor30to60minutes,protectedfromlight.

5.4   MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=360±10/450±10nm.

References&Citations

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Theimpactofvariousscaffoldcomponentsonvascularizedboneconstructs
Authors:AhmadEweida,MatthiasSchulte,OliverFrisch,UlrichKneser,LeilaHarhaus
Journal:JournalofCranio-MaxillofacialSurgery(2017)

AMineralizedHighStrengthandToughHydrogelforSkullBoneRegeneration
Authors:BingXu,PengbinZheng,FeiGao,WeiWang,HongtaoZhang,XuranZhang,XuequanFeng,WenguangLiu
Journal:AdvancedFunctionalMaterials(2016)

DRGaxonelongationandgrowthconecollapserateinducedbySema3AaredifferentlydependentonNGFconcentration
Authors:AndriusKaselis,RimantasTreinys,RutaVosyliute,SauliusSatkauskas
Journal:Cellularandmolecularneurobiology(2014):289--296

Acuteoraltoxicityandkineticbehaviorsofinorganiclayerednanoparticles
Authors:JinYu,Hea-EunChung,Soo-JinChoi
Journal:JournalofNanomaterials(2013):12

EffectofSomeAntihypertensiveDrugsonAlkalinePhosphataseandDNAofMice
Authors:OYEl-Khawaga,AEl-Waseef,YOEllazec,MMEl-Naggar,ABDMAlla
Journal:InternationalJournalofGenomicsandProteomics(2013):60

Anengineeringunderstandingofthesmallintestine
Authors:MonicaRosaliaJaimeFonseca
Journal:(2012)

Cytotoxicityandalkalinephosphataseactivityevaluationofendosequencerootrepairmaterial
Authors:MahmoudRezaModareszadeh,PeterMDiFiore,DavidATipton,NargesSalamat
Journal:Journalofendodontics(2012):1101--1105

Alginate-loadedliposomescanprotectencapsulatedalkalinephosphatasefunctionalitywhenexposedtogastricpH
Authors:AlanMSmith,MonicaRJaime-Fonseca,LiamMGrover,SerafimBakalis
Journal:Journalofagriculturalandfoodchemistry(2010):4719--4724

Regulationoftheosteoblast-specifictranscriptionfactorOsterixbyNO66,aJumonjifamilyhistonedemethylase
Authors:KrishnaMSinha,HideyoYasuda,MadeleneMCoombes,SharonYRDent,BenoitDeCrombrugghe
Journal:TheEMBOjournal(2010):68--79

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