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MICAL(MIC01)isanintracellularflavoproteinmonoxygenase,conservedfrominsectstomammals,thatfunctionsasacatalystforoxidation-reduction(redox)reactions.Terman’sgroupshowedthatMICALinteractswithF-actinandusesNADPHasacofactortooxidizeactinatMet44andMet47.AspartoftheMOXtrue™productline,HumanMICAL1protein(MIC01)[accession#Q8TDZ2(MICA1_HUMAN)]hasbeenproducedandpurifiedfromabacterialexpressionsystem.TherecombinantproteinisN-terminally6xHistidinetaggedandiscomprisedoftheRedoxandCHdomains(aa:1-612)ofthefulllengthprotein.ValidationstudieshaveshownthatMIC01canoxidizeactinwhichwillreducesubtilisinAcleavageby>90%inlimitedproteolysisassays.MIC01hasalsobeenvalidatedfortheproductionofoxidizedactinwhichcanbeusedindownstreamapplicationssuchassedimentationassaysandactinpolymerizationassays.
TolearnmoreaboutusingMICALasaresearchtoolseeourDatasheet
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TolearnmoreabouttheMICAL/MsrB/ActinphysiologicalredoxsystemseeourNewsletter
Eachlotofpurifiedproteinisqualitycontrolledtoprovidehighbatchtobatchconsistency,seeCOAdocuments.
ValidatedApplications
MICAL-1(MIC01)ProteinPurityDetermination A10μgsampleofMICAL-1proteinwassepa-ratedbyelectrophoresisina4-20%Tris-GlycinegelandstainedwithCoomassieBlue.ProteinquantitationwasperformedusingthePrecisionRedTMProteinAssayReagent(Cat.#ADV02).Mark12standardmolecularweightMarkersarefromInvitrogen. |  | |
SubtilisinAssayonMICAL-1TreatedActin Actin(Cat#AKL99)wasdilutedto0.1mg/ml(2.3mM)andleftuntreated,ortreatedwithNADPH(400mM),MIC01(0.076mM),oracombinationofboth(seemethod).2mgofeachsamplewasthenleftuntreated,ortreatedwithsubtilisin(1:200w/w)for15min.SampleswerethenseparatedbySDS-PAGEandvisualizedwithCoomassiestaining. Clickhereforadetailedmethod |  | |
ActinSedimentation+/-MICAL-1Treatment Actin(Cat#AKL99)wasdilutedto0.2mg/ml(4.6mM)or0.8mg/ml(18.4mM)(seemethod).Sampleswerethenincubatedwith2xpolymerizationbufferor2xpolymerizationbuffersupplementedwithMIC01+NADPHfor1hatroomtemperatire.Sampleswerespuninanultracentrifugeat100,000gfor1.5h.SampleswerethenseparatedbySDS-PAGEandvisualizedwithCoomassiestaining. Clickhereforadetailedmethod |  | |
ActinPolymerization+/-MICAL-1Treatment. Pyrenelabeled-actin(Cat.#AP05)wasdilutedto0.1mg/ml(2.3mM)(seemethod).Sampleswerethenincubatedwith2xpolymerizationbuffersupplementedwithnothing(labeledControl),NADPHalone(labeled+NADPH),MIC01alone(labeled+MIC01),orthecombinationofboth(labeled+Both).Uponactinpolymerizationfluorescencewasdetectedwithaspectrophotometer(seemethodbelow).A.U.=arbitraryunits Clickhereforadetailedmethod |  |
