| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 380/464 |
| MW | 278.26 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related | BiochemicalAssays |
1.PrepareFluorescamineworkingsolution:AddthewholecontentofDMSO(ComponentB)intothebottleofFluorescamine(ComponentA),andmixwell.
Note:2.5mLoffluorescamineworkingsolutionisenoughfor1plate.Unusedfluorescamineworkingsolutionshouldbestoredinsingleusealiquotsat-20oCandprotectedfromlight.
2.PrepareBSAstandardsolutions:DilutetheappropriateamountofBSAStandard,1mg/mL(ComponentC)intoPBStogetaBSAconcentrationof100-1.56µg/mLwith2Xdilutions.
Table1LayoutofBSAstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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BS1 | CS1 | …. | …. | …. | …. |
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BS2 | CS2 |
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BS3 | CS3 |
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BS4 | CS4 |
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BS5 | CS5 |
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BS6 | CS6 |
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BS7 | CS7 |
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Note:BS=BSAStandards,BL=BlankControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
BSAStandard | BlankControl | TestSample |
SerialDilutions*:75μL | PBS:75μL | 75μL |
*Note:AddtheserialdilutionsofBSAstandardfrom100to1.56µg/mLwith2XdilutionsintowellsfromBS1toBS7induplicate(seeStep2).
3.Runproteinassay:
3.1 AddtheserialdilutionsofBSAstandardat75μL/well(seeTables1&2)intothe96-wellplate.A0µg/mLBSAcontrolisincludedasblankcontrol.
3.2 Add25μL/welloffluorescamineworkingsolution(fromStep1)intoBSAstandard,blankcontrol,andtestsamples(seeStep2,Table1)tomakethetotalassayvolumeof100µL/well.
Note:Fora384-wellplate,add30μLofsampleand10μLoffluorescamineworkingsolutionintoeachwell.
3.3 Incubatethereactionatroomtemperaturefor5to30minutes,protectedfromlight.
3.4 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=380/470nm.
| References&Citations |  PrinterFriendlyVersion |
1. EastwoodD,FernandezC,YoonBY,SheaffCN,WaiCM.(2006)Fluorescenceofaromaticaminesandtheirfluorescaminederivativesfordetectionofexplosivevapors.ApplSpectrosc,60,958.
2. AdamouR,ColyA,DouabaleSE,SaleckML,Gaye-SeyeMD,TineA.(2005)Fluorimetricdeterminationofhistamineinfishusingmicellarmediaandfluorescamineaslabellingreagent.JFluoresc,15,679.
3. EggenreichK,ZachE,BeckH,WintersteigerR.(2004)Determinationof4-amino-m-cresoland5-amino-o-cresolbyhighperformanceliquidchromatographyandfluorescencederivatizationusingfluorescamine.JBiochemBiophysMethods,61,35.
4. SkelleyAM,MathiesRA.(2003)Chiralseparationoffluorescamine-labeledaminoacidsusingmicrofabricatedcapillaryelectrophoresisdevicesforextraterrestrialexploration.JChromatogrA,1021,191.
5. CesurN,ApakTI,Aboul-EneinHY,OzkirimliS.(2002)LCdeterminationofaminoglutethimideenantiomersasdansylandfluorescaminederivativesintabletformulations.JPharmBiomedAnal,28,487.
6. BelalF,ABDineH,Al-MajedA,KhalilNY.(2002)Spectrofluorimetricdeterminationofvigabatrinandgabapentininurineanddosageformsthroughderivatizationwithfluorescamine.JPharmBiomedAnal,27,253.
7. Bantan-PolakT,KassaiM,GrantKB.(2001)Acomparisonoffluorescamineandnaphthalene-2,3-dicarboxaldehydefluorogenicreagentsformicroplate-baseddetectionofaminoacids.AnalBiochem,297,128.
8. HongC,KondoF.(2000)Detection,quantitation,andidentificationofresidualaminopenicillinsbyhigh-performanceliquidchromatographyafterfluorescaminederivation.JFoodProt,63,1421.
9. KellyMT,FabreH,PerrettD.(2000)Determinationoftaurineinplasmabycapillaryzoneelectrophoresisfollowingderivatisationwithfluorescamine.Electrophoresis,21,699.
10. SeguraCarreteroA,Cruces-BlancoC,FernandezPeinadoS,ElBergmiR,FernandezGutierrezA.(1999)Fluorimetricdeterminationofprocaineinpharmaceuticalpreparationsbasedonitsreactionwithfluorescamine.JPharmBiomedAnal,21,969.
