Host/IsotypeMouse/IgG1 CloneP4D1 SpeciesReactivityWiderangeofspecies

Anti-ubiquitinantibodyisapan-ubiquitin,mousemonoclonalantibodythatispartoftheSignal-Seeker™productline.Theantibodywasraisedagainstfulllengthbovineubiquitin.Theantibodyhasbeenshowntorecognizepoly-ubiquitin,mono-ubiquitin,andfreeubiquitin(Fig1).UbiquitinisahighlyconservedproteinandAUB01ispredictedtorecognizeubiquitinfromawiderangeofspecies(5).AUB01ispurifiedbyProteinGaffinitychromatographyandissuppliedasalyophilizedwhitepowder.

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ValidatedApplications

WesternBlotusingUbiquitinAntibody Anti-UbiquitinAntibody(Cat.#AUB01)wasusedata1:500dilution.Bovinethymusubiquitinwasrunasfollows;Lane1-50ng,Lane2-25ng.Lane3–12.5ng,Lane4-6.25ng,Lane5-3.12ng.Lanes6&7represent20µgofSwiss3T3celllysatefromcellstreatedfor5hwith10µMMG132(Lane6)oruntreatedcells(Lane7).Arrowindicatesfreeubiquitinband(8kD),highermolecularweightbandsareubiquitinatedproteins.ToseethefullWesternblotprotocol,seetheproductdatasheet.

ImmunofluorescenceUbiquitinAntibody HeLacellswerestainedandvisualizedbyfluorescencemicroscopyasdescribedintheIFmethodbelow.UbiquitintargetedcytoplasmicandnuclearproteinsandfreeubiquitinwerestainedusingAUB01at1:500dilution.ToseethefullImmunofluorescenceprotocol,seetheproductdatasheet.

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UbiquitinBackground

Ubiquitin(Ub)andubiquitin-likeproteins(Ubls,e.g.SUMO,Nedd)areagroupofapproximately15proteinsthathaveamolecularweightofaround8kD.Duringtheubiquitinationprocess,theseareconjugatedviaactivating(E1),conjugating(E2)andligating(E3)enzymestolysinesofatargetprotein(1).Mammaliancellsexpressover600potentialubiquitinligaseswhichexceedsthatofthekinasesuperfamilyofPTMproteins(2).

Onefunctionofubiquitinationistotargetproteinsforproteosomaldegradation.Thisrolecanrangefromageneralhousekeepingfunctionthatclearsmissfoldedproteinsfromacelltoinvolvementintightlyregulatedspatio-temporalcellsignalingevents(1).AnemergingfunctionofubiquitinationisitsABIlitytoactivateproteinsviathecreationofuniqueprotein:proteininteractions(3).IncommonwithmanyotherPTMs,ubiquitinationisreversIBLe.Ubiquitin-specificproteases(USPsorDUBs)removeubiquitinsfromtargetproteins(4).ThereversiblenatureofubiquitinationfurtherenhancesthepotentialofthisPTMtodynamicallyregulateproteinfunction.

References

1)Grabbe,C.etal.2011.Thespatialandtemporalorganizationofubiquitinnetworks.Nat.Rev.Mol.Cell.Biol.12:295-307.

2)DeshaiesR.J.&Joazeiro,C.A.2009.RINGdomainE3ligases.Ann.Rev.Biochem.78:399-434.

3)Lomeli,H.&Vazquez.2011.EmergingrolesoftheSUMOpathway.CellMol.LifeSci.68:4045-4064.

4)Faesen,A.C.etal.2012.TheroleofUBLdomainsinubiquitinspecificproteases.Biochem.Soc.Trans.40:539-545.

5)Zuin,A.etal.2014.Ubiquitinsignaling:Extremeconservationasasourceofdiversity.Cells3:690-701.

Formoreinformationcontact:signalseeker@Cytoskeleton.com

AssociatedProducts:

Signal-Seeker™UbiquitinationDetectionKit(Cat.#BK161)

Signal-Seeker™UbiquitinAffinityBeads(Cat.#UBA01-beads)

Signal-Seeker™:BlastR™RapidLysatePrepKit(Cat.#BLR01)

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