| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 405/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection Phosphatases |
| Related | BiochemicalAssays |
1.Prepare100XpNPPstocksolutions:
Add300µLofdistilledH2OintothevialofpNPP(ComponentA).Mixthereagentswell.ThepNPPstocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandrefrozenat-20oC.
Note:Avoidrepeatedfreeze-thawcycles.Thesolutionshouldbegoodfor3-4weeksifstoredat-20oC.
2.PreparepNPPreactionmixture:
PreparepNPPreactionmixtureaccordingtothefollowingtableandkeepfromlight.
Table1.pNPPReactionMixtureforone96-wellplate
Components | Volume |
pNPPstocksolution(100X,fromStep1) | 50µL |
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.Theunusedsolutionshouldbealiquotedandstoredat-20oC.Avoidrepeatedfreeze-thawcycles.
3.2 Add10μLof100units/mLAlkalinePhosphataseStandardsolution(fromStep3.1)to990µLofH2O-0.1%BSAtogeneratea1,000mU/mLAlkalinePhosphataseStandardsolution.
3.3 Take100μLof1,000mU/mLAlkalinePhosphataseStandardsolution(fromStep3.2)toperform1:10andthen1:3serialdilutionstoget100,30,10,3,1,0.3,0.1,and0mU/mLseriallydilutedAlkalinePhosphataseStandards.
3.4 Addseriallydilutedalkalinephosphatasestandardsandalkalinephosphatasecontainingtestsamplesintoawhite/clearbottom96-wellmicroplateasdescribedinTables2and3.
Note1:Preparecellsortissuesamplesasdesired.
Note2:Unusedportionofdilutedalkalinephosphatasestandardsolutionshouldbediscarded.
Table2LayoutofAlkalinephosphatasestandardsandsamplesinawhite/clearbottom96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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AS1 | AS1 | …. | …. | …. | …. |
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AS2 | AS2 |
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AS3 | AS3 |
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AS4 | AS4 |
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AS5 | AS5 |
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AS6 | AS6 |
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AS7 | AS7 |
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Note:AS=AlkalinePhosphataseStandards;BL=BlankControl;TS=TestSamples
Table3Reagentcompositionforeachwell
AlkalinePhosphataseStandards | BlankControl | TestSample |
SerialDilutions*:50μL | H2O-0.1%BSA:50μL | 50μL |
Note:Addtheseriallydilutedalkalinephosphatasestandardsfrom100to0.01mU/mLintowellsfromAS1toAS7induplicate.
4.Runalkalinephosphataseassayinsupernatants:
4.1 Add50μLofassayreactionmixture(fromStep2)intoeachwellofalkalinephosphatasestandard,blankcontrol,andtestsamples(seeStep3.4,Table3)tomakethetotalalkalinephosphataseassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
4.2 Incubatethereactionatthedesiredtemperaturefor10to30minutes,protectedfromlight.
4.3 Monitortheabsorbanceincreasewithanabsorbanceplatereaderat400nm.
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)of1:1dilutedassayreactionmixturetothecellwells(fromStep5.2).
5.3 Incubatethereactionatthedesiredtemperaturefor30to60minutes,protectedfromlight.
5.4 Monitortheabsorbanceincreasewithanabsorbanceplatereaderat400nm.
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