• My NEB
  • Print
  • PDF
  • CDK1-cyclin B

    Description

    CDK1-cyclin B (Cyclin Dependent Kinase 1), also known as Cdc2-cyclin B (Cell division control protein kinase 2) or M-phase promoting factor (MPF), is a member of cyclin-dependent kinases implicated in cell cycle control in eukaryotes. Activation of the CDK1-cyclin B brings the onset of mitosis and is tightly regulated. CDK1-cyclin B is a serine/threonine protein kinase composed of the catalytic subunit CDK1 and its positive regulatory subunit cyclin B (B1 isoform). Binding of CDK1 to cyclin B is essential for activation of the kinase. Phosphorylation of T161 is required for activation of the CDK1-cyclin B complex and is mediated by the CDK activating kinase (CAK). During G2 phase, CDK1-cyclin B complex is held in an inactive state by phosphorylation of CDK1 at the two negative regulatory sites, T14 and Y15 by CDK1 inhibitory protein kinases, Myt1 and Wee1 respectively. Dephosphorylation of T14 and Y15 by cell division cycle (CDC25) protein phosphatase in late G2 phase activates the CDK1-cyclin B complex and triggers the initiation of mitosis. During expression in insect cells, the recombiniant CDK1-cyclin B is activated in vivo by endogenous kinase (1–4).

    Highlights

    • Protein serine/threonine kinase
    • Human, recombinant (S. frugiperda S19)

    Product Source

    Isolated from Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus carrying human CDK1 and human cyclin B (1,2) (kindly provided by Dr. H. Piwnica-Worms).

    Recognition Determinant

    The substrate specificity of CDK1-cyclin B shows an absolute requirement for Pro in the +1 position, a secondary requirement for Arg or Lys at +3, and a preference for basic residues at +2 or +3 positions. The recognition motif for phosphorylation by CDK1-cyclin B is S/TPXR/K, frequently with additional basic residues on either side (K/RSPR/PR/K/H) (5).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer for Protein Kinases (PK)-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of CDK1-cyclin B required to catalyze the transfer of 1 pmol of phosphate to CDK Peptide Substrate PKTPKKAKKL-NH2 (50 μM) in 1 minute at 30°C in a total reaction volume of 30  μl.

    Reaction Conditions

    1X NEBuffer for Protein Kinases (PK)
    Supplement with 200 μM ATP
    Incubate at 30°C

    1X NEBuffer for Protein Kinases (PK):
    50 mM Tris-HCl
    10 mM MgCl2
    0.1 mM EDTA
    2 mM DTT
    0.01% Brij 35
    pH 7.5 @ 25°C

    Storage Temperature

    -70°C

    Storage Conditions

    50 mM HEPES
    100 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.01% Brij 35
    pH 7.5 @ 25°C

    Heat Inactivation

    65°C for 20 min

    Specific Activity

    1,000,000 units/mg

    Storage Notes

    • Avoid repeated freeze/thaw cycles.

    Notes

    1. Molecular Weights
      Theoretical:
      34 kDa (CDK1), 48 kDa (cyclin B)
      Apparent:
      60 kDa on SDS-Page (cyclin B)
    2. Avoid freeze/thaw cycles. Can be stored for 1 week or less at -20°C.
    3. If the source of protein to be phosphorylated is a crude extract of cells or tissue, it is very important to include the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation.
    4. Reaction Conditions for Unit Definition Assay: 1X NEBuffer for PK (NEB #B6022), supplemented with 200 µM ATP (NEB #P0756) and gamma-labeled ATP to a final specific activity of 100-500 µCi/µmol.
    5. Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
    6. If possible, the ATP concentration should be at or near saturation (5 -10-fold over Km). Apparent Km values of ATP for most protein kinases are below 100 μM. 

      However, if the objective is to measure enzyme activity using gamma-labeled ATP, it is best to use 100-200 μM ATP in order to have higher specific activity of gamma-labeled ATP (100-500 cpm/pmol). Also, an excess of substrate should be used, and the level of phosphorylation should not exceed 10% for determination of the initial rate. 

      To phosphorylate a protein or peptide substrate to completion, the ATP concentration should be about 5-fold over the limited substrate concentration. Higher enzyme concentration and prolonged incubation times should be employed. 

      Recommended reference: 
      Protein Phosphorylation: A Practical Approach (1993) ed. Hardie, D.G. IRL Press.

    References

    1. Parker, L.L. et al. EMBO J.. 10
    2. Atherton-Fessler, S. et al. Mol. Cell. Biol.. 13
    3. McGowan, C.H. and Russell, P. The EMBO Journal. 12
    4. Liu, F. et al. Mol. Cell. Biol.. 17
    5. Songyang, Z. et al. Curr. Biol.. 4

    FAQs

    1. Can the histones be used as substrates for protein modification enzymes? Which ones?

    Tech Tips

    This is a single cocktail that combines all five enzymes
    Can be used under native or denaturing conditions
    Under native conditions we recommend adding more enzyme and using longer incubation times
    Enzyme activity in this mix is inhibited by SDS, therefore under denaturing conditions it is essential to have NP-40 present in the reaction mixture in a 1:1 ratio.
    A good positive control substrate is Fetuin.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

    Selection Tools

    Interactive Tools

    Quality Control

    Quality Assurance Statement

    • CDK1-cyclin B contains no detectable protease or phosphatase activities.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Phosphatase Activity (PNPP):
      The product is tested in a reaction containing a p-nitrophenyl phosphate (PNPP), a chromogenic substrate for most phosphatases. After incubation the percent degradation is determined by spectrophotometric analysis of released p-nitrophenol at 405 nm.
    • Protease Activity (SDS-PAGE):
      The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.