标签归档：kinase

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

Casein Kinase II (CK2) |NEB酶试剂 New England Biolabs

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上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

Casein Kinase II (CK2) is a constitutively active serine/threonine protein kinase composed of two 44 kDa catalytic α-subunits and two 26 kDa regulatory β-subunits in an α2β2 configuration to form stable heterotetramers. CK2 holoenzyme undergoes autophosphorylation at two serine residues (S2/S3) of its β-subunit. Recently it has been shown that CK2 α-subunits undergo intermolecular tyrosine-autophosphorylation at Y182, which may represent a specific regulatory mechanism. Also, CK2 is able to phosphorylate, under special circumstances, tyrosyl residues in proteins. CK2 is implicated in a variety of cellular functions (1,2).

产品来源

Isolated from a strain of E. coli expressing both α and β CK2 subunits derived from a human glioblastoma cDNA library (kindly provided by Dr. D. Marshak) (3).

识别决定因素

The CK2 substrate specificity is invariably determined by multiple acidic residues located at positions between -2 and +5 relative to the target amino acid (mostly Ser and rarely Thr). The general recognition motif for phsophorylation by CK2 is SXXE/D, although SXE/D and S/D, and variations of these sequences are also phosphorylated. Polyanionic compounds, like heparin, inhibit CK2 activity with a Ki of 1.4 nm (4,5).

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
    • P6010S     -80    
        Casein Kinase II (CK2) P6010SVIAL -80 1 x 0.02 ml 500,000 units/ml
        NEBuffer™ for Protein Kinases (PK) B6022SVIAL -20 1 x 1 ml 10 X
    • P6010L     -80    
        Casein Kinase II (CK2) P6010LVIAL -80 1 x 0.1 ml 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 CK2 required to catalyze the transfer of 1 pmol of phosphate to CK2 Peptide Substrate, RRRADDSDDDDD (100 µM), in 1 minute at 30°C in a total reaction volume of 25 µl (4,5).

    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)

    存储注意事项

    • Avoid repeated freeze/thaw cycles.

  • 相关产品

    相关产品

    • 5´-三磷酸腺苷（ATP）

    单独销售的组分

    • NEBuffer for Protein Kinases (PK)

  • 注意事项

    1. Molecular Weight: α-subunit (45 kDa), β-subunit (25 kDa). The apparent molecular weight of the α-subunit estimated by SDS-PAGE is about 42 kDa.
    2. General notes:
      • For short term storage (two weeks or less) CK2 can be stored at -20°C. 
      • 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. 
      • Reaction Conditions: 1X NEBuffer for Protein Kinases (PK), supplement with 200 µM ATP and gamma-labeled ATP to a final specific activity of 100-500 µCi/µmol. (NEBuffer for Protein Kinases (PK) will also accept GTP as a phosphoryl donor in place of ATP).
    3. Usage notes:
      • Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate. 
      • 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.

  • 参考文献

    1. Chester, N. and Marshak, D.R. (1993). Anal. Biochem. 209, 284-290.
    2. Donella-Deana, A. et al. (2001). Biochem J. 357, 563-567.
    3. Marin, O. et al. (1999). J. Biol. Chem. 274, 29260-29265.
    4. Marin, O. et al. (1994). BBRC. 198, 898-905.
    5. Sarno, S. et al. (1996). J. Biol. Chem. 271, 10595-10601.

工具 & 资源

  • 选择指南

    • Protein Kinase Substrate Recognition

FAQs & 问题解决指南

  • FAQs

    1. Can you recommend inhibitors for CK2 (P6010)?
    2. How much Casein Kinase II (NEB# P6010) should be used?
    3. What is the consensus sequence for this CK2 (P6010)?

  • 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 SXXE/D

    Heparin inhibits CKII at a Ki of 1.4 nM

T4 Polynucleotide Kinase (3′ phosphatase minus) |NEB酶试剂 New England Biolabs

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上海金畔生物科技有限公司代理New England Biolabs（NEB）酶试剂全线产品，欢迎访问官网了解更多产品信息和订购。

产品信息

T4 Polynucleotide Kinase catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of double- and single-stranded DNA and RNA, as well as nucleoside 3´-monophosphates (1-5). This modified version exhibits full kinase activity with no 3´ phosphatase activity (6,7).

产品来源

An E. coli strain that carries a plasmid encoding the modified T4 Polynucleotide Kinase gene.

产品类别:
Kinases Products,
RNA Modification

应用:
Phosphorylation (Kinase)

  • 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
    • M0236S     -20    
        T4 Polynucleotide Kinase (3′ phosphatase minus) M0236SVIAL -20 1 x 0.02 ml 10,000 units/ml
        T4 Polynucleotide Kinase Reaction Buffer B0201SVIAL -20 1 x 1 ml 10 X
    • M0236L     -20    
        T4 Polynucleotide Kinase (3′ phosphatase minus) M0236LVIAL -20 1 x 0.1 ml 10,000 units/ml
        T4 Polynucleotide Kinase Reaction Buffer B0201SVIAL -20 1 x 1 ml 10 X

  • 特性和用法

    单位定义

    One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of acid-insoluble [32P] in 30 minutes at 37°C (1). 

    Unit Assay Conditions: 
    1X T4 Polynucleotide Kinase Reaction Buffer, 66 µM [γ-32P] ATP (5 x 106 cpm/µmol), 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA.

    反应条件

    1X T4 Polynucleotide Kinase Reaction Buffer
    Incubate at 37°C

    1X T4 Polynucleotide Kinase Reaction Buffer
    70 mM Tris-HCl
    10 mM MgCl2
    5 mM DTT
    (pH 7.6 @ 25°C)

    使用浓度

    10,000 units/ml

    贮存溶液

    10 mM Tris-HCl
    50 mM KCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    0.1 µM ATP
    pH 7.4 @ 25°C

    热失活

    65°C for 20 minutes

    单位活性检测条件

    1X T4 Polynucleotide Kinase Reaction Buffer, 66 µM [γ-32P] ATP (5 x 106 cpm/µmol), 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA.

  • 优势和特性

    Features

    5´ phosphorylation of DNA/RNA for subsequent ligation
    End-labeling of DNA or RNA (2)
    5´ phosphorylation of 3´ phosphorylated mononucleotides to generate a substrate (pNp) that can be added to the 3´ end of DNA or RNA by ligase activity (8)
    5´ end labeling of 3´ phosphorylated oligos (9)

  • 相关产品

    相关产品

    • t1010-monarch-plasmid-miniprep-kit
    • Monarch® DNA 胶回收试剂盒
    • Monarch® PCR & DNA 纯化试剂盒（5 μg）

    单独销售的组分

    • T4 多聚核苷酸激酶反应缓冲液

  • 注意事项

    1. Gaps can be phosphorylated with elevated levels of ATP. Nicks are not phosphorylated efficiently. CTP, GTP, TTP, UTP, dATP or dTTP can be substituted for ATP as a phosphate donor (1).
    2. Dephosphorylation prior to end-labeling can be avoided by utilizing the exchange reaction, although this results in lower specific activity labeling.
    3. Sufficient incorporation levels can be attained using the supplied buffer supplemented with 100 µM ADP in the final reaction. However, higher levels of incorporation with the exchange reaction are achieved when using the buffer described in (2).

  • 参考文献

    1. Richardson, CC. (1981). The Enzymes. 14, 299-314.
    2. Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual. 10.59-10.67,11.31-11.33.
    3. Cameron, V. and Uhlenbeck, O.C. (1977). Biochemistry . 16, 5120-5126.
    4. Berkner, K.L. and Folk, W.R. (1977). J. Biol. Chem.. 252, 3176-3184.
    5. Soltis, D.A. and Uhlenbeck, O.C (1982). J. Biol. Chem.. 257, 11332-11339.
    6. Wang, L.K. and Human, S (2001). J. Biol. Chem.. 276, 26868-26874.
    7. Wang, L.K. and Shuman, S. (2002). Nucl. Acids Res.. 30, 1073-1080.
    8. Vance, JR. and Wilson, T.E. (2001). Mol. Cell. Riot.. 21, 7191-7198.
    9. Interthal, H. et al. (2005). J. Biol. Chem.. 280, 36518-36528.

操作说明、说明书 & 用法

  • 操作说明

    1. Radioactive Labeling with T4 PNK or T4 PNK (3´ phosphatase minus)
    2. Non-radioactive Phosphorylation with T4 PNK or T4 PNK (3´ phosphatase minus)

  • 使用指南

    • Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

FAQs & 问题解决指南

  • FAQs

    1. Will T4 PNK (3′ phosphate minus) work in rCutSmart Buffer?