© 2007 Cell Signaling Technology, Inc. This productisfor Store at –80°C was analyzedusingSDS/PAGE followedbyCoomassiestain. Figure 1.ThepurityoftheGST-CDK1/ A2fusionprotein byCDK/. peptides thatarephosphorylated A consensussequence,(S/T)PX(R/K),isidentifiedinthe CDKs andcyclinsplayaroleinrecognizingsubstrates. of peptidesbyCDK/cyclincomplexessuggeststhatboth in a region called the T loop. Examining the phosphorylation threonine residue of a cyclin conserved and phosphorylation tions. ActivationofCDKsrequiresbindingtoaspecific andprotein-proteininterac regulated byphosphorylation in transcription(1,2).ThekinaseactivityofCDKsistightly and cellcycle.CDK8/CycCCDK9/CycTareinvolved CDK7/CycH isbelievedtoformalinkbetweentranscription regulates neuronmigrationduringbraindevelopment(6). the cellcycle.CDK5isactivatedinpostmitoticneuronsand sively accumulateascellstransitionthroughthisphaseof vated bymitogenicsignalingduringearlyG1andprogres progression (4,5).CDK4/CycDandCDK6/CycDareacti transition whiletheCDK2/CycAcomplexregulatesSphase complexes withcyclinE,whichregulatetheG1-Sphase eton proteinsinvolvedinmitosis.CDK2andCDK3form ofcytoskel /Bandregulatesphosphorylation with oneortwocyclins(1-3).CDK1formsacomplex associates withoneortwoCDKsandmostassociate relocation andmodificationbyotherproteins.Eachcyclin phoshorylation, destruction, relocation,inhibitory/activating these complexesoccursthroughcyclinproductionandits are keyregulatorsinmammaliancellcycle.Regulationof Background: fusion . CDK1 kinaseandCyclinA2protein,suppliedasaGST Description: #7477 n CDK1/Cyclin A2Kinase 3  in vitro 5 µg Purified recombinantfull-lengthhuman Cyclins andcyclin-dependentkinases(CDK) 116 153 212 kD 37 27 61 97 43 56 researchuseonlyandisnotintendedforinhumansoranimals. a CDK1 Cyclin A2 - - - -

A2: variable. Substrate: Histone H1 400 ng/μL, and recombinant CDK1/Cyclin 0.4 mMEDTA, 5mMMgCl MOPS, pH7.2,2.5mM radiometric assayusingthefollowingreaction conditions:5mM Figure 2.CDK1/CyclinA2kinaseactivitywas measuredina Background References: activity wasdeterminedusingaradiometricassay[Fig.2]. lowed byCoomassiestain[Fig.1].CDK1/CyclinA2kinase The purityofthekinasewasassessedusingSDS-PAGE fol lecular weightoftheGST-Cyclin A2fusionproteinis78kDa GST-CDK1 fusionproteinis58kDa.Thetheoreticalmo Quality Control: affinity chromatographyusingGSH-agarose. GST tag.Theproteincomplexwaspurifiedbyone-step Accession No.NM_001237),bothwithanamino-terminal No. NM_001786)andCyclinA2(Met1-Leu432)(GenBank length humanCDK1(Met1-Met297)(GenBankAccession using sf9cellsandrecombinantvirusesencodingfull- were co-expressedusingabaculovirusexpressionsystem Source/Purification:

(6)  (7)  (3)  (1)  CPM (5)  (4)  (2)  100000 125000 150000 175000 25000 50000 75000 Xie, Y. andTsai, L.H.(2004) Holmes, J.K.andSolomon,M.J.(1996) Chow, J.P. etal. (2003) Schang, L.M.(2002) Golsteyn, R.M.(2005) Hofmann, F. andLivingston,D.M. (1996) Murray, A.W. (2004) Chem. 10, 851–861. 40815–40828. 779–792. 0 0 271,25240–25246. 25 The theoreticalmolecularweightofthe 50 β CDK1/Cyclin A2(ng/25µl) The CDK1andCyclinA2proteins -glycerophosphate, 1mMEGTA, 2 , 0.05mMDTT, 50μMATP, Cell J. Antimicrob.Chemother. 75 Kinase activity New 12/07 Cancer Lett. J. Biol.Chem. 116,221–234. 100 125 27 pmol/µgxmin Specific activity 217,129–138. 278, 150 3,108–110. J. Biol. Dev. 175

50,

- 200 -

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: #7477

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

Orders 10 µlHistoneH1(1.0µg/µl),and50.16µCi/µl[ To startthereactioncombine10µldilutedCDK1/CyclinA2kinasesolution, 2-fold serialdilutions. Dilute CDK1/CyclinA2proteinto20ng/µlwith1Xassaybufferfollowedby Transfer enzymefrom-80°Ctoice.Allowthawon Dilute [ Dilute 10mMATP with3Xassaybuffer1:40tomake250µMATP. Histone H1(1.0µg/µl) 32 ATP (10mM)#9804 0.5 mMDTT 50 mMMgCl 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. Protocol forCDK1/CyclinA2KinaseAssay 32 P] ATP solution. 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

Count samplesinascintillationcounter. Transfer P81paperto4mlscintillationtubethenadd3cocktail. theP81paperthenwashwith1%phosphoricacid3times. Air dry onto phosphocelluloseP81paper. After 15minutesterminatereactionbyspotting20µlofthemixture 400 ng/µLHistoneH1 0.05 mMDTT 4 mMMgCl 1 mMEGTA 2.5 mM 5 mMMOPS,pH7.2 b -glycerophosphate

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