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Ex/Em(nm)	646/660
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™AlkalinePhosphataseAssayKitusesaproprietaryflurogenicphosphatasesubstrate,toquantifyalkalinephosphataseactivityinsolutions,incellextractsaswellasonsolidsurfaces(suchasPVDFmembranes).Thisproprietaryflurogenicphosphatasesubstrategeneratesafluorescentproductthathasstronglyredfluorescenceuponinteractionwithphosphatase.Thekitprovidesalltheessentialcomponentswithouroptimized"mixandread"assayprotocolthatiscompatIBLewithHTSliquidhandlinginstruments.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Prepare250XSunRed™Substratestocksolution:

Add100µLofdoublesterileH₂OintothevialofSunRed™Substrate(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbemakesingleusedaliquotsandfrozenat-20^oC.

Note:Avoidrepeatedfreezeandthawcycles.

2.Prepareassayreactionmixture:

Prepareassayreactionmixtureaccordingtothefollowingtableandkeepfromlight.

Table1.Assayreactionmixtureforone96-wellplate(2X)

Note:Preparefreshreactionmixtureforeachexperiment.

3.Prepareseriallydilutedalkalinephosphatasestandards(0to100mU/mL):

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

Note:Thealkalinephosphatasestandardsolutionisnotstable.Unusedstandardsolutionshouldbealiquotedandstoredat-20^oC.Avoidrepeatedfreezeandthawcycles.

3.2   Add10μLof100units/mLalkalinephosphatasestandardsolution(fromStep3.1)to990µLofH₂O-0.1%BSAtogeneratea1,000mU/mLalkalinephosphatasestandardsolution.

3.3   Take100μLof1,000mU/mLalkalinephosphatasestandardsolution(fromStep3.2)toperform1:3serialdilutionstoget300,100,30,10,3,1,0.3,and0mU/mLseriallydilutedalkalinephosphatasestandards.

3.4   Addseriallydilutedalkalinephosphatasestandardsand/oralkalinephosphatasecontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables2and3.

Note:Preparecellsortissuesamplesasdesired.Discardedunusedseriallydilutedalkalinephosphatasestandards.

 

Table2LayoutofAlkalinePhosphataseStandardsandtestsamplesinasolidblack96-wellmicroplate

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

Table3.Reagentcompositionforeachwell:

Note:Addtheseriallydilutedalkalinephosphatasestandardsfrom100to0.01mU/mLintowellsfromAS1toAS7induplicate.

 

4.Runalkalinephosphataseassayinsupernatants:

4.1   Add50μLofassayreactionmixture(fromStep2)toeachwellofalkalinephosphatasestandard,blankcontrol,andtestsamples(seeStep3.4,Table3)tomakethetotalalkalinephosphataseassayvolumeof100µL/well.

Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.

4.2 Incubatethereactionfor30to120minutesatthedesiredtemperature,protectedfromlight.

4.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=620/660nm(cutoff630nm).

 

5.Runalkalinephosphataseassayincells:

5.1   Treatthecellsasdesired.

5.2   Removethegrowthmediumcompletelyfromthecellplate.

Note:ItisimportanttoremovethegrowthmediumcompletelyfromthecellplateduetotheinterferenceofthegrowthmediumwiththeSunRed™Substrate.

5.3   Make1:1dilutionofthe5mLassayreactionmixture(fromStep2,Table1)with5mLdistilledH₂O.

5.4   Add100μL(96-wellplate)or50uL(384-wellplate)of1:1dilutedassayreactionmixture(fromStep5.3)intoeachcellwell(fromStep5.2).

5.5   Incubatethereactionfor30to60minutesatthedesiredtemperature,protectedfromlight.

5.6   MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=620/660nm(cutoff630nm).

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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