| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 650/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection DiagnosticMolecules |
| Related | CellMetabolism BiochemicalAssays |
1. Prepareserialdilutionsofurea(0to1mM)solutions:
1.1 Add1μLof1.0MUreaStandard(ComponentD)to999µLDPBStogenerate1.0mMstandardureasolution.
1.2 Take300μLof1.0mMstandardtoperform1:3serialdilutionstoget300,100,30,10,3,1,and0µMstandardureasolutions.
1.3 Addureastandardsandureacontainingtestsamplesintoa96-wellclearbottommicroplateasdescribedinTables1and2.
Table1.Layoutofureastandardsandtestsamplesinaclearbottom96-wellmicroplate:
BL | BL | TS | TS | …. | …. |
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US1 | US1 | …. | …. | …. | …. |
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US2 | US2 |
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US3 | US3 |
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US4 | US4 |
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US5 | US5 |
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US6 | US6 |
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US7 | US7 |
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Note:US=UreaStandards,BL=BlankControl,TS=TestSamples.
Table2.Reagentcompositionforeachwell:
UreaStandard | BlankControl | TestSample |
Serialdilutions*:50μL | DPBS:50μL | 50μL |
*Note:Addtheseriallydilutedureastandardsfrom1to1000µMintowellsfromUS1toUS7induplicate.
2. PrepareAssayReactionMixtureI:
2.1 Add100µLofddH2OintothevialofAssayEnzymeMix(ComponentA)tomake100XAssayEnzymeMixsolution.
2.2 Add50μLreconstitutedAssayEnzymeMixsolutioninto5mLAssayBufferItomakeAssayReactionMixtureI.
Note1:TheAssayreactionmixtureIshouldbeusedpromptlyandkeptfromlight.Theassaysensitivitywillbedecreasedwithlongerstoragetime.ThefreshAssayEnzymeMixtureIisrecommendedforthebestresult.
Note2:Anyremaining100Xenzymemixsolution(fromStep2.1)needstobealiquotandfrozenat-20°C,avoidrepeatedfreeze-thawcycles.
3. Runureaassay:
3.1 Add50μLofAssayReactionMixtureI(fromStep2.2)toeachwelloftheureastandard,blankcontrol,andtestsamples(seeStep1.3)sothatthetotalassayvolumeis100µL/well.
Note:Fora384-wellplate,add25μLsample,25μLofassayreactionmixtureIperwell.
3.2 Incubatethereactionfor30-60minutesatroomtemperatureor37°C,protectedfromlight.
3.3 Add50μLofAssayBufferII(ComponentC)toeachwellsothatthetotalassayvolumeis150µL/well.
Note:Fora384-wellplate,add25μLAssayBufferII(ComponentC)toeachwell.
3.4 Incubateatroomtemperaturefor10-15minutes,andmonitortheabsorbanceincreaseat660-670nmusinganabsorbancemicroplatereader.
Note1:ThecolorturnstoyellowafterAssayBufferII(ComponentC)isadded,andthewellswithureastandardorsampleswillshowblue-greencolorafterincubation.Theintensityofthecolorwillreachthemaximumin15-30minutes,andisproportionaltotheconcentrationofurea.
Note2:Thefinalcolorisstablefor~1hourinroomtemperatureandthecolorintensitywilldecreasewithtime.
| References&Citations |  PrinterFriendlyVersion |
1. LawsonTS,ConnallyRE,VemulpadS,PiperJA.(2011)Dimethylformamide-free,urea-NaClfluorescenceinsituhybridizationassayforStaphylococcusaureus.LettApplMicrobiol.
2. LiangA,QinH,ZhouL,ZhangY,OuyangH,WangP,JiangZ.(2011)AnovelandsensitiveresonancescatteringassayfordetectionofureainserumcoupledureasecatalyticreactionandNH4+associatedparticlereaction.BioprocessBiosystEng,34,639.
3. FerrucioB,FranchiCA,BoldrinNF,deOliveiraML,deCamargoJL.(2010)Evaluationofdiuron(3-[3,4-dichlorophenyl]-1,1-dimethylurea)inatwo-stagemouseskincarcinogenesisassay.ToxicolPathol,38,756.
4. NgokaLC.(2008)Sampleprepforproteomicsofbreastcancer:proteomicsandgeneontologyrevealdramaticdifferencesinproteinsolubilizationpreferencesofrADIoimmunoprecipitationassayandurealysisbuffers.ProteomeSci,6,30.
5. MulagalapalliS,KumarS,KalathurRC,KayasthaAM.(2007)Immobilizationofureasefrompigeonpea(Cajanuscajan)onagartabletsanditsapplicationinureaassay.ApplBiochemBiotechnol,142,291.
6. RozetE,WascotteV,LecouturierN,PreatV,DeweW,BoulangerB,HubertP.(2007)ImprovementofthedecisionefficiencyoftheaccuracyprofilebymeansofadesirABIlityfunctionforanalyticalmethodsvalidation.Applicationtoadiacetyl-monoximecolorimetricassayusedforthedeterminationofureaintransdermaliontophoreticextracts.AnalChimActa,591,239.
7. KimuraS,IyamaS,YamaguchiY,KanakuraY.(2003)Newenzymaticassayforserumureanitrogenusingureaamidolyase.JClinLabAnal,17,52.
8. FarkasA,VamosR,BajorT,MullnerN,LazarA,HrabaA.(2003)Utilizationoflacrimalureaassayinthemonitoringofhemodialysis:conditions,limitationsandlacrimalarginasecharacterization.ExpEyeRes,76,183.
9. KimuraS,YamanishiH,IyamaS,YamaguchiY,KanakuraY.(2003)Enzymaticassayfordeterminationofbicarbonateioninplasmausingureaamidolyase.ClinChimActa,328,179.
10. BilardiC,BiaginiR,DulbeccoP,IiritanoE,GambaroC,MeleMR,BorroP,TessieriL,ZentilinP,MansiC,VigneriS,SavarinoV.(2002)Stoolantigenassay(HpSA)islessreliablethanureabreathtestforpost-treatmentdiagnosisofHelicobacterpyloriinfection.AlimentPharmacolTher,16,1733.
