Product Pathways - Cell Cycle / Checkpoint
HTScan® CDK1/CycB Kinase Assay Kit #7519
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.
| Kit Includes | Quantity |
|---|---|
| Phospho-Rb (Ser780) Antibody # 9307 | 30 microliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| Rb (Ser780) Biotinylated Peptide # 1142 | 1.25 milliliters |
| CDK1/CycB Kinase # 7518 | 5 micrograms |
Description
The kit provides a means of performing kinase activity assays with recombinant human CDK1/CycB kinase. It includes active CDK1/CycB kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine/threonine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Weights
Peptide substrate, Biotin-Rb (Ser780): 2,150 Daltons. GST-CDK1 kinase: 64 kDa, GST-CycB: 78 kDa.
Peptide Core Sequence
TLS*PI
Kinase Assay - DELFIA
Figure 1. CDK1/CycB kinase activity was measured in a radioisotopic filter binding 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, recombinant CDK1/CycB: 100 ng/50 µl.
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.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition 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, 50 ng CDK1/CycB, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 4. Peptide concentration dependence 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, 50 ng of CDK1/CycB and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 2. Time course 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, 50 ng CDK1/CycB and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human CDK1 (Met1-Met295) (GenBank Accession No. NM_001786) and full-length human CycB (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.
Quality Control
The substrate peptide was selected using our Serine/Threonine Kinase Substrate Screening Kit #7400. Phospho-Rb (Ser780) Antibody #9307 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified CDK1/CycB kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the CDK1/CycB kinase was determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify CDK1/CycB activity using the CDK1/CycB substrate peptide provided in this kit. CDK1/CycB sensitivity to the inhibitor staurosporine was measured using the CDK1/CycB substrate peptide provided in this kit [Fig.5].
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.
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- 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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