| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/560 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection Phosphatases |
| Related | BiochemicalAssays |
1.Prepareassayreactionmixture:
MixthewholecontentofPhosphataseSubstrate(ComponentA)withReactionBuffer(ComponentB)andkeepfromlight.
2.Prepareseriallydilutedalkalinephosphatasestandards(0to100mU/mL):
2.1 Add100μLofdistilledH2Owith0.1%BSA(H2O-0.1%BSA)intothevialofalkalinephosphatasestandard(ComponentC,10units)togeneratea100units/mLalkalinephosphatasestandardsolution.
Note:Thealkalinephosphatasestandardsolutionisnotstable.Unusedsolutionshouldbealiquotedandstoredat-20oC.Avoidrepeatedfreezeandthawcycles.
2.2 Add10μLof100units/mLalkalinephosphatasestandardsolution(fromStep2.1)into990µLofH2O-0.1%BSAtogeneratea1,000mU/mLalkalinephosphatasestandardsolution.
2.3 Take100μLof1,000mU/mLalkalinephosphatasestandardsolution(fromStep2.2)toperform1:100andthen1:3serialdilutionstoget10,3,1,0.3,0.1,0.03,0.01,and0mU/mLseriallydilutedalkalinephosphatasestandards.
2.4 Addseriallydilutedalkalinephosphatasestandardsand/oralkalinephosphatasecontainingtestsamplesintoasolidwhite96-wellmicroplateasdescribedinTables1and2.
Note1:Preparecellsortissuesamplesasdesired.
Note2:Unusedserialdilutionsofalkalinephosphatasestandardshouldbediscarded.
Table1.Layoutofalkalinephosphatasestandardsandtestsamplesinasolidwhite96-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.
Table2.Reagentcompositionforeachwell
AlkalinePhosphataseStandards | BlankControl | TestSample |
SerialDilutions*:50μL | H2O-0.1%BSA:50μL | 50μL |
*Note:Addseriallydilutedalkalinephosphatasestandardsfrom10to0.01mU/mLintowellsfromAS1toAS7induplicate.
3.Runalkalinephosphataseassayinsupernatants:
3.1 Add50μLofassayreactionmixture(fromStep1)intoeachwellofalkalinephosphatasestandard,blankcontrol,andtestsamples(seeStep2.4,Table1)tomakethetotalalkalinephosphataseassayvolumeof100µL/well
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
3.2 Incubatethereactionfor30to60minutesatroomtemperature,protectedfromlight.
3.3 Add50μLofAssayBuffer(ComponentD)intoeachwellofalkalinephosphatasestandard,blankcontrol,andtestsampleswithassayreactionmixture(seeStep3.2)tomakethetotalalkalinephosphataseassayvolumeof150µL/well
Note:Fora384-wellplate,add25μLofAssayBuffer(ComponentD)intoeachwell.
3.4 Incubatethereactionfor10to30minutesatroomtemperature,protectedfromlight.
3.5 Monitortheluminescenceincreasewithastandardluminescenceplatereader.
4.Runalkalinephosphataseassayincells:
4.1 Treatthecellsasdesired.
4.2 Removethegrowthmediumcompletelyfromthecellplate.
Note:Itisimportanttoremovethegrowthmediumcompletelyfromthecellplateduetotheinterferenceofthegrowthmediumwiththephosphatasesubstrate.
4.3 Make1:1dilutionofthe5mLassayreactionmixture(fromStep1)with5mLdistilledH2O.
4.4 Add100μL(96-wellplate)or50uL(384-wellplate)of1:1dilutedassayreactionmixture(fromStep4.3)intothecellwells(fromStep4.2).
4.5 Incubatethereactionatthedesiredtemperaturefor30to60minutes,protectedfromlight.
4.6 Add50μL(96-wellplate)or25uL(384-wellplate)ofAssayBuffer(ComponentD)intothecellwellscontainingassayreactionmixture(fromStep4.5).
4.7 Incubatethereactionatroomtemperaturefor10to30minutes,protectedfromlight.
4.8 Monitortheluminescenceincreasewithastandardluminescenceplatereader.
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