© 2005 Cell Signaling Technology, Inc. This productisfor Store at -80°C SDS/PAGE followedbyCoomassiestain. Figure 1.ThepurityoftheIRAK2proteinwasanalyzedusing activated (3). cascades,whichincludeTAK1, IKKsandthestress- is thenreleasedintothecytoplasmandactivatesprotein TAB2 andTRAF6.TheresultingIRAK-TRAF6-TAB2 complex and itsassociationwithtwomembrane-boundproteins: dissociation fromtheIL-1RIcomplex,itsubiquitination after IL-1stimulation.ThesubsequenteventsinvolveIRAK shortly and IRAKs(2).IRAKundergoesautophosphorylation the formation of a complex that includes IL-1RI, AcP, MyD88 The bindingofIL-1toreceptortypeI(IL-1RI)initiates contains fourmembers(IRAK1,IRAK2,IRAK-MandIRAK4). receptor (1).ThemammalianfamilyofIRAKmolecules be coprecipitatedinanIL-1-induciblemannerwiththeIL-1 kinase (IRAK)isaserine/threonine-specificthatcan Background: protein. IRAK2 (Met1-Pro625)kinase,suppliedasaGSTfusion Description: #7367 n IRAK2 Kinase 3  in vitro 5 µg Purified recombinantfull-lengthhuman Interleukin-1 (IL-1)receptor-associated researchuseonlyandisnotintendedforinhumansoranimals. 116 212 kD 158 27 35 66 97 43 56 a IRAK2

5 mMMgCl pH 7.2, 2.5 mM assay usingthefollowingreactionconditions:5mMMOPS, Figure 2.IRAK2kinaseactivitywasmeasuredinaradiometric Background References: Molecular Formula: using aradiometricassay[Fig.2]. massie stain[Fig.1].IRAK2kinaseactivitywasdetermined controlled forpurityusingSDS-PAGE followedbyCoo IRAK2 proteinis103kDa.Thepurifiedkinasewasquality Quality Control: chromatography usingglutathione-agarose. minal GSTtag.Theproteinwaspurifiedbyone-stepaffinity (GenBank AccessionNo.NM_001570)withanamino-ter with aconstructexpressinghumanIRAK2(Met1-Pro625) was producedusingabaculovirusexpressionsystem Source/Purification: MBP 200ng/μl,andvariableamountsofrecombinantIRAK2.

CPM (2)  (1)  (3)  10000 20000 30000 40000 50000 60000 70000 80000 Takaesu, G.etal.(2001) Dinarello, C.A.(1996) Janssens, S.andBeyaert,R.(2003) 293–302. 2475–2484. 0 0 2 , 0.05mMDTT, 50μMATP, Substrate: β -glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 100 The theoreticalmolecularweightofthe 103000 The GST-Kinase fusionprotein 200 IRAK2 (ng/25µl) Kinase activity Blood New 01/08 Mol. Cell.Biol. 300 87,2095–2147. 20 pmol/µgxmin Specific activity 400 Mol. Cell 21, 500

11, - 600

- 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 ATP (10mM)#9804 Support Orders issuppliedin50mMTris-HCl, pH7.5; Web n n n www.cellsignal.com [email protected] 877-678-TECH (8324) [email protected] 877-616-CELL (2355)

page  of 2 © 2005 Cell Signaling Technology, Inc. B A lot ofkinaseunderspecifiedconditions. assay incubationtimesandenzymeconcentrationsmustbedeterminedempiricallyforeach Note:

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

Orders MBP (0.5µg/µl),and5µl0.16µCi/µl[32]ATP solution. To startthereactioncombine10µldilutedIRAK2kinasesolution, followed by2-foldserialdilutions. Dilute IRAK2protein(100ng/µlconcentration)to60with1Xassaybuffer Transfer enzymefrom-80°Ctoice.Allowthawon Dilute [ Dilute 10mMATP with3Xassaybuffer1:40tomake250µMATP. MBP (0.5µg/µl) 32 ATP (10mM)#9804 0.25 mMDTT 25 mMMgCl 2 mMEDTA 5 mMEGTA 12.5 mM 25 mMMOPS,pH7.2 Kinase Buffer(5X) P- g ATP 32 n p] ATP to0.16µCi/µl[ b 877-616-CELL (2355)[email protected] -glycerophosphate

2

32 p] ATP with250µMATP solution. Protocol for Support

IRAK2 Kinase 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/µlMBP 50 µMATP 0.05 mMDTT 5 mMMgCl 1 mMEGTA 2.5 mM 5 mMMOPS,pH7.2 Assay b -glycerophosphate

2

Web n www.cellsignal.com

page  of 2