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CDK1/Cyclin A2 Kinase CDK1/Cyclin A2 Kinase 3 Store at –80°C n 5 µg Orders n 877-616-CELL (2355) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com New 12/07 #7477 This product is for in vitro research use only and is not intended for use in humans or animals. Description: Purified recombinant full-length human Source/Purification: The CDK1 and Cyclin A2 proteins Storage: Enzyme is supplied in 50 mM Tris-HCl, pH7.5; CDK1 kinase and Cyclin A2 protein, supplied as a GST were co-expressed using a baculovirus expression system 150 mM NaCl, 0.25 mM DTT, 0.1mM EGTA, 0.1 mM EDTA, fusion protein. using sf9 cells and recombinant viruses encoding full- 0.1 mM PMSF, 25% glycerol, 7 mM glutathione. length human CDK1 (Met1-Met297) (GenBank Accession Store at -80°C. Background: Cyclins and cyclin-dependent kinases (CDK) No. NM_001786) and Cyclin A2 (Met1-Leu432) (GenBank are key regulators in mammalian cell cycle. Regulation of Keep on ice during use. Accession No. NM_001237), both with an amino-terminal these complexes occurs through cyclin production and its GST tag. The protein complex was purified by one-step Avoid repeated freeze-thaw cycles. destruction, relocation, inhibitory/activating phoshorylation, affinity chromatography using GSH-agarose. relocation and modification by other proteins. Each cyclin Companion Products: associates with one or two CDKs and most CDKs associate Quality Control: The theoretical molecular weight of the Kinase Buffer (10X) #9802 with one or two cyclins (1-3). CDK1 forms a complex with GST-CDK1 fusion protein is 58 kDa. The theoretical mo- cyclin A/B and regulates phosphorylation of cytoskel- lecular weight of the GST-Cyclin A2 fusion protein is 78 kDa ATP (10 mM) #9804 eton proteins involved in mitosis. CDK2 and CDK3 form The purity of the kinase was assessed using SDS-PAGE fol- Serine/Threonine Kinase Substrate Screening Kit #7400 complexes with cyclin E, which regulate the G1-S phase lowed by Coomassie stain [Fig.1]. CDK1/Cyclin A2 kinase transition while the CDK2/CycA complex regulates S phase activity was determined using a radiometric assay [Fig.2]. progression (4,5). CDK4/CycD and CDK6/CycD are acti- Background References: vated by mitogenic signaling during early G1 and progres- sively accumulate as cells transition through this phase of (1) Schang, L.M. (2002) J. Antimicrob. Chemother. 50, the cell cycle. CDK5 is activated in postmitotic neurons and 779–792. regulates neuron migration during brain development (6). (2) Murray, A.W. (2004) Cell 116, 221–234. CDK7/CycH is believed to form a link between transcription (3) Chow, J.P. et al. (2003) J. Biol. Chem. 278, and cell cycle. CDK8/CycC and CDK9/CycT are involved 40815–40828. in transcription (1,2). The kinase activity of CDKs is tightly regulated by phosphorylation and protein-protein interac- (4) Hofmann, F. and Livingston, D.M. (1996) Genes Dev. tions. Activation of CDKs requires binding to a specific 10, 851–861. cyclin and phosphorylation of a conserved threonine residue (5) Golsteyn, R.M. (2005) Cancer Lett. 217, 129–138. in a region called the T loop. Examining the phosphorylation of peptides by CDK/cyclin complexes suggests that both (6) Xie, Y. and Tsai, L.H. (2004) Cell Cycle 3, 108–110. CDKs and cyclins play a role in recognizing substrates. (7) Holmes, J.K. and Solomon, M.J. (1996) J. Biol. A consensus sequence, (S/T)PX(R/K), is identified in the Chem. 271, 25240–25246. peptides that are phosphorylated by CDK/cyclins. kDa Kinase activity 175000 212 150000 153 116 125000 97 100000 Cyclin A2 CPM 61 75000 50000 56 CDK1 Specific activity 25000 43 27 pmol/µg x min 0 37 0 25 50 75 100 125 150 175 200 CDK1/Cyclin A2 (ng/25 µl) 27 Figure 2. CDK1/Cyclin A2 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl , 0.05 mM DTT, 50 μM ATP, Figure 1. The purity of the GST-CDK1/Cyclin A2 fusion protein 2 Substrate: Histone H1 400 ng/μL, and recombinant CDK1/Cyclin was analyzed using SDS/PAGE followed by Coomassie stain. A2: variable. of 2 1 © 2007 Cell Signaling Technology, Inc. © 2007 Cell Signaling Technology, page Protocol for CDK1/Cyclin A2 Kinase Assay #7477 Note: Lot-specific information for this kinase is provided on the enzyme vial. Optimal Final Assay Conditions assay incubation times and enzyme concentrations must be determined empirically for each 5 mM MOPS, pH 7.2 lot of kinase under specified conditions. 2.5 mM β-glycerophosphate 1 mM EGTA A Additional Solutions and Reagents (Not included) 4 mM MgCl2 0.05 mM DTT 1. Kinase Buffer (10X) 400 ng/µL Histone H1 50 mM MOPS, pH 7.2 25 mM β-glycerophosphate 6. After 15 minutes terminate reaction by spotting 20 µl of the reaction mixture 10 mM EGTA onto phosphocellulose P81 paper. 4 mM EDTA 7. Air dry the P81 paper then wash with 1% phosphoric acid 3 times. 8. Transfer P81 paper to 4 ml scintillation tube then add 3 ml scintillation cocktail. 50 mM MgCl2 0.5 mM DTT 9. Count samples in a scintillation counter. 2. ATP (10 mM) #9804 3. 32P-gATP 4. Histone H1 (1.0 µg/µl) Cell Signaling Technology offers a full line of protein kinases, substrates, and B Suggested Protocol antibody detection reagents for high throughput screening. Please direct all inquiries to: [email protected]. 1. Dilute 10 mM ATP with 3X assay buffer 1:40 to make 250 µM ATP. 2. Dilute [32p] ATP to 0.16 µCi/µl [32p] ATP with 250 µM ATP solution. 3. Transfer enzyme from -80°C to ice. Allow enzyme to thaw on ice. 4. Dilute CDK1/Cyclin A2 protein to 20 ng/µl with 1X assay buffer followed by 2-fold serial dilutions. 5. To start the reaction combine 10 µl diluted CDK1/Cyclin A2 kinase solution, 10 µl Histone H1 (1.0 µg/µl), and 5 µl 0.16 µCi/µl [32P] ATP solution. of 2 2 Orders n 877-616-CELL (2355) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com © 2007 Cell Signaling Technology, Inc. © 2007 Cell Signaling Technology, page .
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