Home Protein Tools cAMP-dependent Protein Kinase (PKA), catalytic subunit

cAMP-dependent Protein Kinase (PKA), catalytic subunit
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The catalytic subunit of cAMP-dependent Protein Kinase (PKA) is a serine/threonine protein kinase. The recognition motif for phosphorylation by PKA is RRXS/TY, where Y tends to be a hydrophobic residue.

  • Recombinant enzyme with no detectable protease or phosphatase contaminating activities
  • Optimal activity and stability for up to 12 months
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Catalog # Cat # Concentration Conc Size List Price List Price Your Price Quantity
P6000S 2,500,000 units/ml 100,000 units $193.00 $193.00
P6000L 2,500,000 units/ml 500,000 units $769.00 $769.00
Please enter a quantity for at least one size
  • The catalytic subunit of cAMP-dependent Protein Kinase (PKA) is a serine/threonine protein kinase, which combines, in the absence of cAMP, with the regulatory subunit to form the inactive PKA holoenzyme. Since this is the free catalytic subunit alone, no cAMP is required for activation (1,2). 

    When purified from mammalian tissue, the PKA catalytic subunit is always phosphorylated at T197, essential for catalysis. Most likely a heterologous kinase is responsible for this in vivo phosphorylation of PKA. Although the fully active PKA expressed in E. coli autophosphorylates on both T197 and S338, this does not reflect the mechanism used in eukaryotic cells (3). 

    Product Source

    Isolated from a strain of E. coli that carries a clone expressing the murine PKA catalytic subunit (α isoform) under control of a T7 expression system (2,3) (cDNA kindly provided by Dr. G.S. McKnight).

    Recognition Determinant

    The recognition motif for phosphorylation by PKA is RRXS/TY, where Y tends to be a hydrophobic residue. A Phe in the nearby sequence tends to be a negative determinant for phosphorylation by PKA. Some variations with regard to spacing and basic residues are permissible (2,4).
    This product is related to the following categories:
    Protein Kinases
    This product can be used in the following applications:
    Protein Phosphatases and Kinases

    • Reagents Supplied

      The following reagents are supplied with this product:

      NEB # Component Name Component # Stored at (°C) Amount Concentration
      • P6000S     -20    
          cAMP-dependent Protein Kinase (PKA), catalytic subunit P6000SVIAL -20 1 x 0.04 ml 2,500,000 units/ml
          NEBuffer™ for Protein Kinases (PK) B6022SVIAL -20 1 x 1 ml 10 X
      • P6000L     -20    
          cAMP-dependent Protein Kinase (PKA), catalytic subunit P6000LVIAL -20 1 x 0.2 ml 2,500,000 units/ml
          NEBuffer™ for Protein Kinases (PK) B6022SVIAL -20 1 x 1 ml 10 X

    • Properties & Usage

      Unit Definition

      One unit of enzyme catalyzes the phosphorylation of 1 pmol of LRRASLGQLTQEEE substrate in 20 minutes at 30˚C.

      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 EGTA
      2 mM DTT
      0.01% Brij 35
      (pH 7.5 @ 25°C)

      Storage Buffer

      20 mM Tris-HCl
      50 mM NaCl
      2 mM DTT
      1 mM EDTA
      50% Glycerol
      pH 7.5 @ 25°C

      Heat Inactivation

      65°C for 20 minutes

      Molecular Weight

      Theoretical: 38 kDa

    • Related Products

      Companion Products

    • Product Notes

      1. Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.
      2. 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.
      3. This clone has a nucleotide sequence identical to the GenBank entry NM_008854, as entered by Dr. G. S. McKnight.
      4. 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.

    • References

      1. Uhler, M.D., Carmichael, D.F., Lee, D.C., Chrivia, J.C., Krebs, E.G. and McKnight, G.S. (1986). Proc.Natl. Acad. Sci. USA. 83, 1300-1304.
      2. Slice, L.W. and Taylor, S.S. (1989). J. Biol. Chem. 264, 20940-20946.
      3. Moore, M.J. et al. (2002). J. Biol. Chem. 277, 47878-47884.
      4. Zetterqvist, O.Z. et al. (1990). B.E. Kemp, ed(Ed.), in Peptides and Protein Phosphorylation. 171-187. CRC Press, Inc. Boca Raton.

    • FAQs

      1. How much cAMP-dependent Protein Kinase (NEB# P6000) should be used?
      2. What is the consensus sequence for PKA (P6000)?

    • Tech Tips

      Make sure to use the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation if the substrate is a crude lysate.
      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.
      1-2 µl of enzyme is a good starting point for a 1 hr incubation of 1µg of protein.
      The consensus sequence is RRXS/TY, where Y tends to be a hydrophobic residue

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