cAMP-dependent Protein Kinase (PKA), catalytic subunit |NEB酶试剂 New England Biolabs

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产品信息

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). 

产品来源

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).

识别决定因素

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).

产品类别:
Protein Kinases Products

应用:
Protein Phosphatases and Kinases

  • 产品组分信息

    本产品提供以下试剂或组分：

    NEB # 名称 组分货号 储存温度 数量 浓度
    • 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

  • 特性和用法

    单位定义

    One unit is defined as the amount of PKA catalytic subunit required to catalyze the transfer of 1 pmol of phosphate to Kemptide, LRRASLG (100 µM) in 1 minute at 30°C in a total reaction volume of 25μL.

    反应条件

    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)

    贮存溶液

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

    热失活

    65°C for 20 minutes

    分子量

    理论上的: 38 kDa

  • 相关产品

    相关产品

    • 5´-三磷酸腺苷（ATP）

  • 注意事项

    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.

  • 参考文献

    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.

工具 & 资源

  • 选择指南

    • Protein Kinase Substrate Recognition

FAQs & 问题解决指南

  • FAQs

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

  • 实验技巧

    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