Product Pathways - Cell Cycle / Checkpoint
CDK1/CycB Kinase #7518
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
Description
Purified recombinant full length human CDK1/CycB kinase, supplied as a GST fusion protein.
Source / Purification
Source/Purification: The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full length human CDK1 (Met1-Met297) (GenBank Accession No. NM_001786) and full length human Cyclin B (Met1-Val433) (GenBank Accession No. NM_031966), both with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-CDK1/CycB fusion protein was analyzed using SDS/PAGE followed by anti-CDK1 (A) and anti-CycB Western blots (B) or Coomassie stain (C).
Kinase Assay - Radiometric
Figure 2. CDK1/CycB kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: Rb CTF, 5 µg /50 µl, and 100 ng/50 µl Recombinant CDK1/CycB.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of CDK1/CycB kinase activity: DELFIA® data generated using Phospho-Rb (Ser780) Antibody #9307 to detect phosphorylation of substrate peptide (#1142) by CDK1/CycB kinase. In a 50 µl reaction, increasing amounts of CDK1/CycB and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Quality Control
The theoretical molecular weight of the GST-CDK1 fusion protein is 64 kDa. The theoretical molecular weight of the GST-Cyclin B fusion protein is 78 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain and Western blot [Fig.1]. CDK1/CycB kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure CDK1/CycB activity using HTScan® CDK1/CycB Kinase Assay Kit #7519 [Fig.3].
Background
Cyclins and cyclin-dependent kinases (CDK) are key regulators in mammalian cell cycle. Regulation of these complexes occurs through cyclin production and its destruction, relocation, inhibitory/activating phoshorylation, relocation and modification by other proteins. Each cyclin associates with one or two CDKs and most CDKs associate with one or two cyclins (1-3). CDK1 forms a complex with cyclin A/B and regulates phosphorylation of cytoskeleton proteins involved in mitosis. CDK2 and CDK3 form complexes with cyclin E, which regulate the G1-S phase transition while the CDK2/CycA complex regulates S phase progression (4,5). CDK4/CycD and CDK6/CycD are activated by mitogenic signaling during early G1 and progressively accumulate as cells transition through this phase of the cell cycle. CDK5 is activated in postmitotic neurons and regulates neuron migration during brain development (6). CDK7/CycH is believed to form a link between transcription and cell cycle. CDK8/CycC and CDK9/CycT are involved in transcription (1,2). The kinase activity of CDKs is tightly regulated by phosphorylation and protein-protein interactions. Activation of CDKs requires binding to a specific cyclin and phosphorylation of a conserved threonine residue in a region called the T loop. Examining the phosphorylation of peptides by CDK/cyclin complexes suggests that both CDKs and cyclins play a role in recognizing substrates. A consensus sequence, (S/T)PX(R/K), is identified in the peptides that are phosphorylated by CDK/cyclins.
- Schang, L.M. (2002) J. Antimicrob. Chemother. 50, 779-792.
- Murray, A.W. (2004) Cell 116, 221-234.
- Chow, J.P. et al. (2003) J. Biol. Chem. 278, 40815-40828.
- Hofmann, F. and Livingston, D.M. (1996) Genes Dev. 10, 851-861.
- Golsteyn, R.M. (2005) Cancer Lett. 217, 129-138.
- Xie, Y. and Tsai, L.H. (2004) Cell Cycle 3, 108-110.
- Holmes, J.K. and Solomon, M.J. (1996) J. Biol. Chem. 271, 25240-25246.
Application References
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Companion Products
- 7519 HTScan® CDK1/CycB Kinase Assay Kit
- 1142 Rb (Ser780) Biotinylated Peptide
- 9307 Phospho-Rb (Ser780) Antibody
- 9802 Kinase Buffer (10X)
- 9804 ATP (10 mM)
- 9953 Staurosporine
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
