CDK6/CyclinD1, His-tag, GST-tag Recombinant

Catalog #

40097

$410 *

Size: 10 µg

Qty

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Description

Recombinant human CDK6, full length, containing an N-terminal His-tag. Recombinant human cyclin D1 (CCND1), full length, containing an N-terminal GST-tag. The two proteins were co-expressed in Sf9 insect cells and spontaneously formed a complex, which was affinity purified.

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Synonyms

CDK6: cyclin dependent kinase 6, Cell division protein kinase 6, PLSTIRE, MCPH12
Cyclin D1: CCND1, B-cell lymphoma 1 protein, BCL1, BCL-1, PRAD1, U21B31, D11S287E, CDK-6

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

Specific Activity

Product Info

Storage and Usage

Citations

Species

Human

Host Species/Expression System

Sf9 insect cells

Purity

≥80%

Format

Aqueous buffer solution

Formulation

50 mM Sodium Phosphate, pH 7.0, 300 mM NaCl, 150 mM imidazole, 0.1 mM PMSF, 0.2 mM DTT, 25% glycerol

CDK6: 40 kDa; CyclinD1: 61 kDa

Amino Acids

CDK6: Full Length; CyclinD1: Full Length

Specific Activity

1.7 pmol/min/µg

Genbank #

CDK6: NM_001259; CyclinD1: NM_053056

UniProt #

CDK6: Q00534; CyclinD1: P24385

Tag(s)

N-terminal His-tag (CDK6), N-terminal GST-tag (CyclinD1)

Background

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 selectivity in vitro; CDK6 phosphorylates Thr821 while CDK4 phosphorylates Thr826 on Rb protein (2).

References

1. Meyerson, M. et al: Identification of G1 kinase activity for cdk6, a novel cyclin D partner. Molec. Cell. Biol. 14: 2077-2086, 1994.
2. Takaki, T. et al: Preferences for phosphorylation sites in the retinoblastoma protein of D-type cyclin-dependent kinases, Cdk4 and Cdk6, in vitro. J Biochem. 2005 Mar;137(3):381-6.

Storage/Stability

At least 6 months at -80°C.

Assay Conditions

The activity of the complex was measured using ADP-Glo™ Kinase Assay kit (Promega; Cat# V9101) which quantifies the amount of ADP produced by phosphorylation. The ADP-Glo™ Reagent is added to terminate the reaction and quench the remaining ATP. The Kinase Detection Reagent is then added to convert ADP to ATP and to measure the newly converted ATP using a luciferase reaction.
Assay: Kinase activity was measured using an Rb protein substrate. The reaction was initiated by mixing increasing amounts of the protein kinase with 25 µM ATP in 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/ml BSA, 50 µM fresh DTT, and Rb substrate (0.04 mg/ml final). The reaction was terminated by addition of an equal volume of the ADP-Glo™ Reagent supplemented with 10mM MgCl2, and the Kinase Detection Reagent was added. Phosphorylation was measured by detection of luminescence. The blank was determined from a “no kinase” sample.

Instructions for Use

Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage. Avoid multiple freeze/thaw cycles.

Applications

Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.

Shipping Temperature

-80°C

Warnings

Avoid freeze/thaw cycles

Scientific Category

Kinase/Serine-Threonine

Regulatory Status

Research Use Only

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Datasheets/Protocols

40097 Lot 211207

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CDK6/CyclinD1, His-tag, GST-tag Recombinant

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