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AAT Bioquest/PhosphoWorks™ Fluorimetric Phosphate Assay Kit *Red Fluorescence*/21660/125 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection Anions |
| Related | ATPases Phosphoproteins |
| Spectrum | AdvancedSpectrumViewer |
Note:Toachievethebestresults,it’sstronglyrecommendedtousetheblackplates.
1.PreparePhosphateSensorworkingsolution:
1.1 Thawoneofeachkitcomponentatroomtemperaturebeforeuse.
1.2 Make125XPhosphateSensorstocksolution:Add20μLofDMSOintoPhosphateSensor(ComponentB)tomake125XPhosphateSensorstocksolution.
1.3 MakePhosphateSensorworkingsolution:Transfer20μLof125XPhosphateSensorstocksolution(fromStep1.2)into2.5mLofsterileH2O,andmixwell
Note1:AvoiddirectexposureofPhosphateSensor(ComponentB)tolight.
Note2:AliquotandstoretheunusedAssayBuffer(ComponentA)andPhosphateSensorstocksolution(fromStep1.2)at-20oC.Avoidrepeatedfreeze/thawcyclesandpotentialPicontamination.
Note3:DuetothehighsensitivityofthisassaytoPi,itisextremelyimportanttousePi-freelaboratorywareandreagents.
2.Prepareseriallydilutedphosphatestandardsandtestsamples:
2.1 Preparephosphatestandard:Add50μLof1mMKH2PO4(ComponentC)into950μLofdeionizedwaterorenzymereactionbuffertogeta50μMphosphatestandardsolution.Thentake200μLof50μMphosphatestandardsolutiontoperform1:2serialdilutionstoget25,12.5,6.25,3.125,1.56,and0.78μMseriallydilutedphosphatestandards.
2.2 Addphosphatecontainingsamplesandseriallydilutedphosphatestandardsintoasolidblack96-wellmicroplateaccordingtoTables1and2.
Table1Layoutofphosphatestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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PS1 | PS1 | …. | …. | …. | …. |
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PS2 | PS2 |
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PS3 | PS3 |
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PS4 | PS4 |
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PS5 | PS5 |
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PS6 | PS6 |
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PS7 | PS7 |
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Note:PS=PhosphateStandard,BL=BlankControl,TS=TestSample.
Table2Reagentcompositionforeachwell
PhosphateStandards | BlankControl | TestSample |
SerialDilutions*:40μL | H2OorBuffer:40μL | 40μL |
Note:*Addtheserialdilutionsofphosphatestandardfrom0.1μMto50μMintowellsfromPS1toPS7.
3.RunPhosphoWorksTMfluorimetricphosphateassay:
Warning:RunthephosphateassayatpH6.5to7.4.
3.1 Add40μLofAssayBuffer(ComponentA)and20µLofPhosphateSensorworkingsolution(fromStep1.3)intoeachwellofphosphatestandard,blankcontrol,andtestsamples(seeStep2.2)tomakethetotalphosphateassayvolumeof100µL/well.
Note:Fora384-wellplate,add20μLofsample,20μLofAssayBuffer(ComponentA)and10µLofPhosphateSensorworkingsolution(fromStep1.3)intoeachwell.
3.2 Incubatethereactionmixtureatroomtemperaturefor15minutesto1hour.
3.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm.
| References&Citations |  CitationExplorer |
Sacrificialcrystaltemplatingofhyaluronicacid-basedhydrogels
Authors:RichelleCThomas,PaulEChung,ShanPModi,JohnGHardy,ChristineESchmidt
Journal:EuropeanPolymerJournal(2016)
Osteogeniccellculturescannotutilizeexogenoussourcesofsyntheticpolyphosphateformineralization
Authors:MarianneBAriganello,SidneyOmelon,FABIoVariola,RimaMWazen,PierreMoffatt,AntonioNanci
Journal:Journalofcellularbiochemistry(2014):2089--2102
