Recombinant full-length human CDK6 and CyclinD3 were co-expressed by baculovirus in Sf9 insect cells using an N-terminal His tag on both proteins. CDK6 is a member of the cyclin-dependent family of protein kinases that are important regulators of cell cycle progression. CDK6 activity is regulated by the D-type cyclins and members of the INK4 family of CDK inhibitors (1). The CDK6 kinase activity is detected in mid-G1 phase of the cell cycle and is responsible for the phosphorylation and regulation of the activity of tumor suppressor protein Rb. Although CDK6 and CDK4 can both phosphorylate multiple residues in the Rb protein, they do so with different residue selectivities in vitro; CDK6 phosphorylates Thr821 while CDK4 phosphorylates Thr826 on Rb protein (2).
ADP-Glo™ Kinase Assay is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is a substrate in a reaction catalyzed by Ultra-Glo™ Luciferase that produces light. The luminescent signal positively correlates with ADP amount and kinase activity. The assay is well suited for measuring the effects chemical compounds have on the activity of a broad range of purified kinases, making it ideal for both primary screening as well as kinase selectivity profiling. The ADP-Glo™ Kinase Assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) using up to 1mM ATP.
Profile More Compounds In-House: ADP-Glo™ Kinase Assay + Kinase Enzyme System is optimized so that you are up and running in no time. Complete Systems: The Kinase Enzyme Systems include a recombinant kinase enzyme, a substrate appropriate for the enzyme, a reaction buffer, DTT and supplemental reagents as needed. Obtain Reliable Results: The broad dynamic range, the ease of use and better sensitivity obtained with ADP-Glo™ Kinase Assay result in less ambiguous data.
Notes
Kinase Enzyme System manufactured by SignalChem.
Bulk quantities available upon request.
References
1.Meyerson, M. et al. (1994) Identification of G1 kinase activity for cdk6, a novel cyclin D partner. Molec. Cell. Biol. 14, 2077–86.
2.Takaki, T. et al. (2005) Preferences for phosphorylation sites in the retinoblastoma protein of D-type cyclin-dependent kinases, Cdk4 and Cdk6, in vitro. J Biochem. 137, 381–6.