© 2006 Cell Signaling Technology, Inc. This productisfor Store at -80°C analyzed usingSDS/PAGE followedbyCoomassiestain. Figure 1.ThepurityoftheGST-MYLK2 fusionprotein was from smoothmuscle(4). myosinlightchainsisolated specifically phosphorylates or smoothmuscle.Incontrast,muscleMYLK myosinlightchainsisolatedfromskeletal phosphorylate insmoothmuscletissue(3).SkeletalMYLKcan observed more ubiquitouslyexpressedwiththegreatestexpression twitch skeletalmuscles(2).ThesmoothmuscleMYLKis tension potentiationduetorepetitivestimulationinfast- expressed predominantlyinskeletalmusclesandregulates there are2genesforMYLK.TheskeletalMYLK(MYLK2)is permeability andmusclecontraction(1).Invertebrates including cellspreadingandmigration,transepithelial to playmajorrolesinanumberofbiologicalprocesses, the actin-activatedmyosinATPase activityandisthought light increases chain (RLC). This regulatory phosphorylation ofmyosin myosin motoractivitiesthroughphosphorylation Ca Background: protein. MYLK2 (Met1-Val596) kinase,suppliedasaGSTfusion Description: #7498 2+ /calmodulin-dependent proteinkinasesthatregulate n Kinase MYLK2 3  in vitro 5 µg Purified recombinantfulllengthhuman Myosin lightchainkinases(MYLKs)are 100 150 250 kDa researchuseonlyandisnotintendedforinhumansoranimals. 15 20 25 37 50 75

MYLK2 GST Background References: determined usingaradiometricassay[Fig.2]. by Coomassiestain[Fig.1].MYLK2kinaseactivitywas was qualitycontrolledforpurityusingSDS-PAGE followed GST-MYLK2 fusionproteinis95kDa.Thepurifiedkinase Quality Control: step affinitychromatographyusingGSH-agarose. amino-terminal GSTtag.Theproteinwaspurifiedbyone- Val596) (GenBankAccessionNo.NM_033118.2)withan construct expressingfulllengthhumanMYLK2(Met1- produced usingabaculovirusexpressionsystemwith Source/Purification:

recombinant MYLK2:200ng/50μl. derived peptide(R11-SGRARTSSFAEPGGK) 200ng/μL, and 2.5 μg/50μlPEG20,000,ATP variable,Substrate:GSK3 3 μMNa-orthovanadate,1.2mMDTT, 0.04mg/mlcalmodulin, NaOH, pH7.5,3mMMgCl assay usingthefollowingreactionconditions:60mMHEPES- Figure 2.MYLK2kinaseactivitywasmeasuredinaradiometric (1)  (4)  (3)  (2)  Kamm, K.E.andStull,J.T. (2001) Herring, B.P. etal.(1992) Lazar, V. andGarcia, J.G.(1999) Zhi, G.etal.(2005) V (pmol ATP/µg x min) 25945–25950. 256–267. 17519–17524. 4527-4530. 15 25 20 10 0 5 1 0 n = 4 The theoreticalmolecularweightofthe 0 The GST-Kinase fusionproteinwas Proc. Natl.Acad.Sci.USA 2 , 3mMMnCl 20 New 11/06 Kinase activity Conc. AT V max J. Biol.Chem. P- K =20pmol/µgxmin A m 30 TP (µM) =6.1µM Genomics J. Biol.Chem 2 , 0.4mMCaCl 40 267, 57, 102, 50 . 276, 2 ,

Kinase Buffer(10X)#9802 Serine/Threonine KinaseSubstrateScreeningKit#7400 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: ATP (10mM)#9804 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: #7498

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

Orders 5 µl0.16µCi/µl[ derived peptide(500ng/µL),2µL0.5mg/mlcalmodulinin5mMCaCl To startthereactioncombine10µldilutedMYLK2kinasesolution,GSK3 serial dilutions. Dilute MYLK2kinaseproteinto20ng/µlwith1Xassaybufferfollowedby2-fold Transfer enzymefrom-80°Ctoice.Allowthawon Dilute [ Dilute 10mMATP with3Xassaybuffer1:40tomake250µMATP. GSK3 derivedpeptide(R11-SGRARTSSFAEPGGK) (500ng/µL) 0.5 mg/mlcalmodulinin5mMCaCl 32 ATP (10mM)#9804 12.5 µg/50µlPEG 6 mMDTT 15 µMNa-orthovanadate 15 mMMnCl 15 mMMgCl 300 mMHEPES-NaOH,pH7.5 Kinase Buffer(5X) P- g ATP 32 n p] ATP to0.16µCi/µl[ 877-616-CELL (2355)[email protected]

2 2

32 20,000 p] ATP solution.

32 p] ATP with250µMATP solution. 2 Protocol forMYLK2KinaseAssay Support 2 , and 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 200 ng/µLGSK3derivedpeptide 0.04 mg/mlcalmodulin 50 ng/µlPEG 1.2 mMDTT 3 µMNa-orthovanadate 0.4 mMCaCl 3 mMMnCl 3 mMMgCl 60 mMHEPES-NaOH,pH7.5 2 2 20,000 2 Web n www.cellsignal.com

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