| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 560/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | NeuroBIOLOGy ReactiveOxygenSpecies |
| Related | RedoxEnzymes BiochemicalAssays |
1. PrepareserialSOD(0to100U/mL)standardsolutions:
1.1 Add50µLAssayBuffer(ComponentE)intothevialofSODStandard(ComponentD)tomake10kU/mLSODstocksolution.
Note:TheunusedSODsolutionshouldbedividedassingleusealiquotsandstoredat-20°C.
1.2 Add10μLof10kU/mLSODsolutionin990μLofAssayBuffer(ComponentE)toget100U/mLSODsolution.Take100μLof100U/mLSODsolutiontoperform1:10serialandthen1:3dilutionstoget10,3,1,0.3,0.1,0.03U/mLSODstandardssolutions.
1.3 AddSODstandardsandSOD-containingtestsamplesintoa96-wellwhitewall/clearbottomorclearmicroplateasdescribedinTables1and2.
Table1.LayoutofSODstandardsandtestsamplesinaclear96-wellmicroplate:
BL | BL | TS | TS | …. | …. |
|
|
|
|
|
|
SOD1 | SOD1 | …. | …. | …. | …. |
|
|
|
|
|
|
SOD2 | SOD2 |
|
|
|
|
|
|
|
|
|
|
SOD3 | SOD3 |
|
|
|
|
|
|
|
|
|
|
SOD4 | SOD4 |
|
|
|
|
|
|
|
|
|
|
SOD5 | SOD5 |
|
|
|
|
|
|
|
|
|
|
SOD6 | SOD6 |
|
|
|
|
|
|
|
|
|
|
SOD7 | SOD7 |
|
|
|
|
|
|
|
|
|
|
Note:SOD=SuperoxideDismutaseStandards,BL=BlankControl,TS=TestSamples.
Table2.Reagentcompositionforeachwell:
SODStandard | BlankControl | TestSample |
Serialdilutions*(50μL) | Assaybuffer(ComponentE):50μL | 50 μL |
Note: Addtheseriallydilutedsuperoxidedismutasestandardsfrom0.03U/mLto100U/mLintowellsfromSOD1toSOD7induplicate.
2. PrepareSODassaymixture1:
2.1 Add2.5mLAssayBuffer(ComponentE)tothebottleofReadiView™SOD560(ComponentA),mixthemwell.
2.2 Add50µLof50XXanthine(ComponentB)intothebottleofComponentA(fromStep2.1)tomakeSODassaymixture.
Note:TheSODassaymixtureshouldbepreparedjustbeforetheexperiment,andkeptfromlight. Theassaymixtureisnotstableandtheunusedportionshouldbediscarded.
3. PrepareSODassaymixture2:
3.1 Add50µLAssayBuffertothevialofXanthineOxidase(ComponentC),andtransfer50µLXanthineOxidasestocksolutioninto2.5mLAssayBuffer(ComponentE)tomakeSODassaymixture2,mixwell.
4. RunSODassay:
4.1 Add25μLofSODassaymixture1(fromStep2.2)intoeachwelloftheSODstandard,blankcontrol,andtestsamples(seeStep1,Tables1and2).
4.2 Add25μLofSODassaymixture2(fromStep3)intoeachwelloftheSODstandard,blankcontrol,andtestsamples(fromStep4.1).
Note:For384-wellplate,add25μLofsample,12.5μLofSODassaymixture1,and12.5μLofSODassaymixture2intoeachwell.
4.3 Incubateatroomtemperaturefor30-60minutes.
4.4 Monitortheabsorbanceintensity550to560nm
| References&Citations |  PrinterFriendlyVersion |
1. AgnihotriR,PandurangP,KamathSU,GoyalR,BallalS,ShanbhogueAY,KamathU,BhatGS,BhatKM.(2009)Associationofcigarettesmokingwithsuperoxidedismutaseenzymelevelsinsubjectswithchronicperiodontitis.JPeriodontol,80,657.
2. AhlIM,JonssonBH,TibellLA.(2009)ThermodynamiccharacterizationoftheinteractionbetweentheC-terminaldomainofextracellularsuperoxidedismutaseandheparinbyisothermaltitrationcalorimetry.Biochemistry,48,9932.
3. AkalinFA,BaltaciogluE,AlverA,KarabulutE.(2009)Totalantioxidantcapacityandsuperoxidedismutaseactivitylevelsinserumandgingivalcrevicularfluidinpregnantwomenwithchronicperiodontitis.JPeriodontol,80,457.
4. AndradeJrDR,AndradeDR,SantosSA.(2009)Studyofrathepatocytesinprimaryculturesubmittedtohypoxiaandreoxygenation:actionofthecytoprotectorsprostaglandinE1,superoxidedismutase,allopurinolandverapamil.ArqGastroenterol,46,333.
5. Assadpoor-PiranfarM,PordalAH,BeyranvandMR.(2009)Measurementofoxidizedlow-densitylipoproteinandsuperoxidedismutaseactivityinpatientswithhypertension.ArchIranMed,12,116.
6. AzeemiST,RazaSM,YasinzaiM,SamadA.(2009)Effectofdifferentwavelengthsonsuperoxidedismutase.JAcupunctMeridianStud,2,236.
7. BalasubramanianV,EzhevskayaM,MoonsH,NeuburgerM,CristescuC,VanDoorslaerS,PalivanC.(2009)Structuralcharacterizationofahighlyactivesuperoxide-dismutasemimic.PhysChemChemPhys,11,6778.
8. BallalA,MannaAC.(2009)Regulationofsuperoxidedismutase(sod)genesbySarAinStaphylococcusaureus.JBacteriol,191,3301.
9. BaoY,LiL,XuF,ZhangG.(2009)Intracellularcopper/zincsuperoxidedismutasefrombayscallopArgopectenirradians:itsgenestructure,mRNAexpressionandrecombinantprotein.FishShellfishImmunol,27,210.
10. ChanJM,OhWK,XieW,ReganMM,StampferMJ,KingIB,AbeM,KantoffPW.(2009)Plasmaselenium,manganesesuperoxidedismutase,andintermediate-orhigh-riskprostatecancer.JClinOncol,27,3577.
