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7522
HTScan® CDK2/CycA Kinase Assay Kit
Cellular Assay Kits
Assay Kit

HTScan® CDK2/CycA Kinase Assay Kit #7522

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Figure 1. CDK2/CycA 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: Histone H1, 1 µg /50 µl, recombinant CDK2/CycA: 50 ng/50 µl.
Figure 3. Dose dependence curve of CDK2/CycA kinase activity: DELFIA® data generated using Phospho-Rb (Ser807/811) Antibody #9308 to detect phosphorylation of substrate peptide (#1144) by CDK2/CycA kinase. In a 50 µl reaction, increasing amounts of CDK2/CycA and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Staurosporine inhibition of CDK2/CycA kinase activity: DELFIA® data generated using Phospho-(Ser807/811) Antibody #9308 to detect phosphorylation of substrate peptide (#1144) by CDK2/CycA kinase. In a 50 µl reaction, 50 ng CDK2/CycA, 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.)
Figure 4. Peptide concentration dependence of CDK2/CycA kinase activity: DELFIA® data generated using Phospho-Rb (Ser807/811) Antibody #9308 to detect phosphorylation of substrate peptide (#1144) by CDK2/CycA kinase. In a 50 µl reaction, 50 ng of CDK2/CycA and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. Time course of CDK2/CycA kinase activity: DELFIA® data generated using Phospho-Rb (Ser807/811) Antibody #9308 to detect phosphorylation of substrate peptide (#1144) by CDK2/CycA kinase. In a 50 µl reaction, 50 ng CDK2/CycA and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Inquiry Info.# 7522

Product Description

The kit provides a means of performing kinase activity assays with recombinant human CDK2/CycA kinase. It includes active CDK2/CycA 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 Formula Peptide substrate, Biotin-Rb (Ser807/811): GSK-CDK2: 60kDa, GST-CycA: 75 kDa.

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.

Pathways

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