© 2007 Cell Signaling Technology, Inc. This productisfor Store at –80°C using SDS/PAGE followedbyCoomassiestain. Figure 1.Thepurityof the AuroraAfusionproteinwasanalyzed Aurora Clocalizesonthecentrosomefromanaphaseto duringtheG2/Mphasetransition (4,5). H3 phosphorylation Aurora AandBistightlycoordinatedwithhistone a varietyofhumancancers(2,4).Theexpressionboth cytokinesis. AuroraBoverexpressionisalsodetectedin the controlofmicrotubule-kinetochoreattachmentand Aurora Bregulateschromosomesegregationthrough prophase andthenrelocalizestothespindleatanaphase. of mitosis.AuroraBassociateswithchromosomesduring activity peaksatthetransitionfrommetaphasetoend during theG2/Mphaseofcellcycleandkinase cancers (2,4).TheexpressionofAuroraBalsopeaks in humanbreast,bladder, colon,ovarianandpancreatic and stability. OverexpressionofAuroraAhasbeendetected centrosome separation,maturationandspindleassembly domain increaseskinaseactivity. AuroraAisinvolvedin ofThr288initscatalytic the cellcycle.Phosphorylation G1 andSphasespeakduringtheG2/Mphaseof spindle andincytoplasm.Itsproteinlevelsarelowduring at thecentrosomes,alongmicrotubulesofmitotic (3). AuroraAisdetectedinmitoticallyproliferatingcells and segregation,cleavagefurrowpositioningingression such ascentrosomeduplication,chromosomebiorientation cytokinesis andmaybeinvolvedinkeycellcycleevents Their functionalinfluencesspanfromG2throughto mitotic cellssuggestanassociationwithstructure. expression andsubcellularlocalizationofAurorakinasesin and AuroraC(1,2).Studiesonthetemporalpatternof members identifiedamongmammals:AuroraA, B family ofmitoticserine/threoninekinaseswiththree Background: protein. Aurora A(Met1-Ser403)kinase,suppliedasaGSTfusion Description: #7388 n Kinase Aurora A 3  in vitro 5 µg 20.0 34.6 66.4 97.2 42.7 55.6 27.0 116 158 212 kDa Purified recombinantfull-lengthhuman Aurora kinases belong to a highly conserved Aurora kinasesbelongtoahighlyconserved researchuseonlyandisnotintendedforinhumansoranimals. Aurora

A 200 ng/μL,andrecombinantAuroraA:variable. EDTA, 5mM MgCl pH 7.2,2.5mM assay usingthefollowingreactionconditions: 5mMMOPS, Figure 2.AuroraAkinaseactivitywasmeasured inaradiometric Background References: determined usingaradiometricassay[Fig.2]. by Coomassiestain[Fig.1].AuroraAkinaseactivitywas quality controlledforpurityusingSDS-PAGE followed Aurora Afusionproteinis72kDa.Thepurifiedkinasewas Quality Control: one-step affinitychromatographyusingGSH-agarose. an amino-terminalGSTtag.Theproteinwaspurifiedby (Met1-Ser403) (GenBankAccessionNo.NM_003600)with with aconstructexpressingfull-lengthhumanAuroraA was producedusingabaculovirusexpressionsystem Source/Purification: cell lines(6). overexpression ofAuroraCisdetectedinvariouscancer of AuroraCshowsthatitsexpressionislimitedtothetestis, peaks duringG2/Mphase.Althoughthetissuedistribution cytokinesis andexpressionofbothmRNAproteinlevels

(1)  (5)  (4)  (3)  (2)  CPM (6)  100000 200000 300000 400000 Warner, S.L.etal.(2003) Crosio, C.etal.(2002) Pascreau, G.etal.(2003) Andrews, P.D. etal.(2003) Katayama ,H.etal.(2003) Kimura, M.etal.(1999) 7334–7340. 369–374. 672–683. 451–464. 589–595. 0 0 β -glycerophosphate, 1mMEGTA, 0.4mM 2 , 0.05mMDTT, 50μMATP, Substrate:MBP The theoreticalmolecularweightofthe 100 The GST-Kinase fusionprotein Aurora A(ng/25µl) Kinase activity New 05/07 Mol. Cell.Biol. J. Biol.Chem. Mol. CancerTher. Prog. CellCycleRes. 200 Cancer MetastasisRev. Curr. Opin.CellBiol. 73 pmol/µgxmin Specific activity 300 22,874–885. 274, 2, 5, 15, 400 22,

ATP (10mM)#9804 Kinase Buffer(10X)#9802 Companion Products: Avoid repeatedfreeze-thawcycles. Keep oniceduringuse. Store at–80°C. 0.1 mMPMSF, 25%glycerol,7mMglutathione. 150 mMNaCl,0.25DTT, 0.1mMEGTA, 0.1mMEDTA, Storage: Serine/Threonine KinaseSubstrateScreeningKit#7400 Support Orders Enzyme issuppliedin50mMTris-HCl, pH7.5; Web n n n www.cellsignal.com [email protected] 877-678-TECH (8324) [email protected] 877-616-CELL (2355)

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B A lot ofkinaseunderspecifiedconditions. assay incubationtimesandenzymeconcentrationsmustbedeterminedempiricallyforeach Note: #7388

5. 4. 3. 2. 1. 4. 3. 2. 1. Suggested Protocol Additional SolutionsandReagents(Notincluded) Lot-specific informationforthiskinaseisprovidedontheenzymevial.Optimal

Orders (0.5 µg/µl),and5µl0.16µCi/µl[ To startthereactioncombine10µldilutedAuroraAkinasesolution,MBP dilutions. Dilute AuroraAproteinto20ng/µlwith1Xassaybufferfollowedby2-foldserial Transfer enzymefrom-80°Ctoice.Allowthawon Dilute [ Dilute 10mMATP with3Xassaybuffer1:40tomake250µMATP. MBP (0.5µg/µl) 32 ATP (10mM)#9804 0.5 mMDTT 50 mMMgC1 4 mMEDTA 10 mMEGTA 25 mM 50 mMMOPS,pH7.2 Kinase Buffer(10X) P- g ATP 32 b n p] ATP to0.16µCi/µl[ -glycerophosphate 877-616-CELL (2355)[email protected]

2

32 p] ATP with250µMATP solution. 32 p] ATP solution. Protocol forAuroraAKinaseAssay Support n

877-678-TECH (8324)[email protected]

to: [email protected]. antibody detectionreagentsforhighthroughputscreening.Pleasedirectallinquiries Cell SignalingTechnology offersafulllineofproteinkinases,substrates,and 9. 8. 7. 6. Final AssayConditions

5 mMMgCl 0.4 mMEDTA 1 mMEGTA Count samplesinascintillationcounter. Transfer P81paperto4mlscintillationtubethenadd3cocktail. theP81paperthenwashwith1%phosphoricacid3times. Air dry onto phosphocelluloseP81paper. After 15minutesterminatereactionbyspotting20µlofthemixture 200 ng/µLMBP 0.05 mMDTT 2.5 mM 5 mMMOPS,pH7.2 b -glycerophosphate 2 Web n www.cellsignal.com

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