| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 316/456 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection Anions |
| Related | CellSignalingMolecules BiochemicalAssays |
1.Prepareassaysolution:
1.1 Thawallthefourcomponentsatroomtemperaturebeforeuse.
1.2 Prepare200XPPiSensorStockSolution:Add50µLofDMSO(ComponentD)intothevialofPPiSensor(ComponentB)tomake200XPPiSensorStockSolution.
Note:25µLofthePPiSensorStockSolutionisenoughforone96-wellplate.TheunusedPPiSensorStockSolutionshouldbedividedintosingle-usealiquots.Storeat-20oCandprotectfromlight.
1.3 PrepareAssaySolution:Add25µLof200XPPiSensorStockSolution(fromStep1.2)to5mLofAssayBuffer(ComponentA),andmixthemwell.
Note1:DuetothehighsensitivityofthisassaytoPPi,itisimportanttousePPi-freelabwareandreagents.
Note2:DTT>1mMwillincreasethebackground,MgCl2>2mMdecreasetheresponse.
2.Prepareseriallydilutedpyrophosphatestandardsandtestsamples:
2.1 Prepare1mMPyrophosphateStandardSolution:Add10μLof50mMPyrophosphateStandard(ComponentC)into490μLofAssayBuffer(ComponentA),orbufferofyourchoice(preferably50mMHepesbuffer,pH7)tomake1mMpyrophosphatestandardsolution.
2.2 Add50μLof1mMpyrophosphatestandardsolution(fromStep2.1)into450μLofAssayBuffer(ComponentA)toget100μMpyrophosphatestandardsolution,andthentake200μLof100μMpyrophosphatestandardsolutiontoperform1:3serialdilutionstoget30,10,3,1,0.3,0.1and0 μMseriallydilutedpyrophosphatestandards.
2.3 Addseriallydilutedpyrophosphatestandardsand/orpyrophosphate-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Table1Layoutofpyrophosphatestandardsandtestsamplesinasolidblack96-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=PyrophosphateStandard,BL=BlankControl,TS=TestSample.
Table2Reagentcompositionforeachwell
PyrophosphateStandards | BlankControl | TestSample |
SerialDilutions*:50μL | AssayBuffer:50μL | 50μL |
Note:*Addseriallydilutedpyrophosphatestandardsfrom0.3μMto100μMintowellsfromPS1toPS7.
3.Runpyrophosphateassay:
3.1 Add50μL/wellofAssaySolution(fromStep1.3)tothewellsofpyrophosphatestandards,blankcontrol,andtestsamples.Mixthereagentsthoroughly.
Note:Fora384-wellplate,add25μLofsampleand25μLofAssaySolutionintoeachwell.
3.2 Incubateatroomtemperaturefor10to30minutes.
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