| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 652/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection MetalIons |
| Related | DiagnosticMolecules BiochemicalAssays |
AssayProtocol
1. Calciumstandardsandtestsamplepreparations:
1.1 Take10μLof300mMCalciumStandardSolution(ComponentC)to990µLDilutionBuffer(ComponentB)toget3
mMCalciumstandardsolution.
1.2 Add50μLof3mMCalciumStandardsolution(fromStep1.1)to950µLDilutionBuffer(ComponentB)toget150µM
Calciumstandardsolution.
1.3 Take500μLof150µMCalciumStandardSolution(fromStep1.2)toperform1:2serialdilutionstoget75,37.5,18.75,
9.375,4.68,2.34and0µMseriallydilutedCa2+standards.
1.4 Dilutethetestsampleto5-100µMrangewithDilutionBuffer(ComponentB).
1.5 Add50μLofCalciumStandard,dilutedsamplesandblankcontrolintoeachwellasshowninTable1.
Table1.Layoutofcalciumstandardsandtestsamplesinaclearbottom96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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CS1 | CS1 | …. | …. | …. | …. |
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CS2 | CS2 |
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CS3 | CS3 |
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CS4 | CS4 |
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CS5 | CS5 |
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CS6 | CS6 |
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CS7 | CS7 |
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Note:CS=CalciumStandards,BL=BlankControl,TS=TestSamples.
Table2.Reagentcompositionforeachwell
CalciumStandard | BlankControl | TestSample |
Serialdilutions*(50μL) | DilutionBuffer(50μL) | 50μL |
*Note:Addtheseriallydilutedcalciumstandardsfrom150µMto2.34µMintowellsfromCS1toCS7induplicate.
2. Runcalciumassay:
2.1 Add50μLofCalciumBlue™(ComponentA)toeachwellofcalciumstandards,blankcontrol,andtestsamples
(seeStep1.5,Table1and2)tomakethetotalcalciumassayvolumeto100µL/well.
Note: Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
2.2 Incubatethereactionfor5-10minutesatroomtemperature,protectedfromlight.
2.3 Measuretheabsorbanceintensitiesat600nmor650nm.
3. Calciumcalibrationcurve:
Plottheassaydataasshownbelow.
Figure1.Calciumdoseresponsewasmeasuredona96-wellblackwall/clearbottomplatewiththeAmplite™Colorimetric
CalciumQuantitationKit.Aslowas~2.5µMCa2+wasdetectedwith5minutesincubationtime(n=3).
AssayProtocolforSerumorUrineSamples
1. Preparecalciumstandardsandtestsamplepreparations:
1.1 Take10μLof300mMCalciumStandardsolution(ComponentC)to990µLDilutionBuffer(ComponentB)toget3mMCalciumStandardSolution.
1.2 Take500μLof3mMCalciumStandardSolutiontoperform1:2serialdilutionstoget1.5,0.75,0.375,0.1875,0.094,0.047and0µMseriallydilutedCa2+standards.
1.3 Add10μLofcalciumstandard,serumorurinesamplesandblankcontrolintoeachwellasshowninTable3.
Table3.Layoutofcalciumstandardsandtestsamplesinaclearbottom96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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CS1 | CS1 | …. | …. | …. | …. |
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CS2 | CS2 |
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CS3 | CS3 |
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CS4 | CS4 |
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CS5 | CS5 |
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CS6 | CS6 |
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CS7 | CS7 |
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Note:CS=CalciumStandards,BL=BlankControl,TS=TestSamples.
Table4.Reagentcompositionforeachwell
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