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AAT Bioquest/Amplite™ Fluorimetric Alkaline Phosphatase Assay Kit *Near Infrared Fluorescence*/11954/500 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 646/660 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection Phosphatases |
| Related | BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Prepare250XSunRed™Substratestocksolution:
Add100µLofdoublesterileH2OintothevialofSunRed™Substrate(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionshouldbemakesingleusedaliquotsandfrozenat-20oC.
Note:Avoidrepeatedfreezeandthawcycles.
2.Prepareassayreactionmixture:
Prepareassayreactionmixtureaccordingtothefollowingtableandkeepfromlight.
Table1.Assayreactionmixtureforone96-wellplate(2X)
Components | Volume |
250XSunRed™Substratestocksolution(fromStep1) | 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.Unusedstandardsolutionshouldbealiquotedandstoredat-20oC.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
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.
Table3.Reagentcompositionforeachwell:
AlkalinePhosphataseStandards | BlankControl | TestSample |
Serialdilutions*:50μL | H2O-0.1%BSA:50μL | 50μL |
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).
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