| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 330/None |
| MW | 313.34 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | SmallMoleculeDetection Anions |
| Related | CellSignalingMolecules BiochemicalAssays |
1.Prepareassayreagents:
1.1 Thawallthefourcomponentsatroomtemperaturebeforeuse.
1.2 PrepareMESGSubstrate(ComponentB)Solution:Add500μLofddH2OtothevialofMESGSubstrate(ComponentB).MixwellbyvortexingtogetMESGSubstrateSolution.
Note:250ulisenoughforoneplatemakesingleusedaliquosandstoreitat-20°CImmediately.,
1.3 PreparePurineNucleosidePhosphorylase(ComponentC)Solution:Add100μLofddH2OtothevialofPurineNucleosidePhosphorylase(PNP;ComponentC).MixwellbyvortexingtogetPurineNucleosidePhosphorylaseSolution.
1.4 PrepareAssaySolution:AddthewholevolumeofMESGSubstrateSolution(fromStep1.2)andPurineNucleosidePhosphorylaseSolution(fromStep1.3)intothebottleofAssayBuffer(ComponentA),mixwelltogettheassaysolution.Placetheassaysolutiononice.
Note1:ThisAssaySolutionisstableforatleast4hoursonice.Itisnotrecommendedtofreezetheassaysolutionforanotherassay.
Note2:Toachievethedesirableresults,UV-transparentplatesorcuvettesarerequired.
Note3:DuetothehighsensitivityofthisassaytoPi,itisextremelyimportanttousePi-freelaboratorywareand
2.Prepareseriallydilutedphosphatestandardsand/ortestsamples:
2.1 PreparePhosphateStandard:Add50μLof1mMKH2PO4(ComponentD)into950μLofdeionizedwaterorenzymereactionbuffertogive50μMphosphatestandardsolution.
2.2 Take200μLof50μMphosphatestandardsolutiontoperform1:2serialdilutionstogive25,12.5,6.25,3.125,1.56,and0.78μMseriallydilutedphosphatestandards.
2.3 Addphosphate-containingtestsamplesand/orphosphatestandardsintoaclearUV-transparent96-wellmicroplateaccordingtoTables1and2.
Table1LayoutofphosphatestandardsandtestsamplesinaclearUV-transparent96-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
PhosphateStandard | BlankControl | TestSample |
SerialDilutions*:50μL | Phosphate-freewaterorbuffer:50μL | 50μL |
*Note:Addtheserialdilutionsofphosphatestandardfrom0.1μMto50μMintowellsfromPS1toPS7.
3.RunPhosphoWorks™MESGphosphateassay:
3.1 Add50μL/wellofAssaySolution(fromStep1.4)intothewellsofphosphatestandards,blankcontrol,andtestsamples.Mixthereagentsthoroughly.
Note:Fora384-wellplate,add25μLofsampleand25μLofAssaySolutionintoeachwell.
3.2 Incubateatroomtemperaturefor30minutes.Monitortheabsorbancewithamicroplatereaderorspectrophotometerat360nm.
Note:Forcuvetteassaythatrequiresthetotalvolumelargerthan100μL,multiplethevolumeofsampleandassayreagentproportionallybeforemeasuringtheabsorption.
| 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
