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

8-Fluo-cAMP


8-Fluo-cAMP

品牌:Biolog
CAS No.:293296-57-8
储存条件:-20℃
纯度:>95% HPLC
产品编号

(生产商编号)

等级 规格 运输包装 零售价(RMB) 库存情况 参考值

BLG-F002-01

1 µmol 咨询


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cAMP/Epac信号通路研究用试剂

  • 产品特性
  • 相关资料
  • Q&A
  • 参考文献

cAMP/Epac信号通路研究用试剂cAMP/Epac信号通路研究用试剂

Biolog总部位于德国,专门从事信号转导研究的工具。已通过 ISO 9001认证,专注于核苷酸化学,特别强调基于环核苷酸的相应蛋白激酶、磷酸二酯酶和离子通道等的调节剂。

 

Epac ,即cAMP直接活化交换蛋白(Exchange protein directly activated by cyclic AMP),是一种新型的 cAMP 受体。

Epac 在许多组织中表达,例如脑、血管、肾脏、肾上腺和胰腺。在大量研究中,Epac 被证实在多种生物功能和过程中发挥作用。近期研究表明 Epac还参与多种神经元功能的调节,但神经系统中依赖于 Epac 的 cAMP 信号的机制尚未完全了解。

Biolog可提供多款cAMP/Epac信号通路研究用试剂。

 


◆特异性 Epac激活剂

产品名称/结构式

产品编号

产品规格

用途

8-Br-2'-O-Me-cAMP

cAMP/Epac信号通路研究用试剂

B 022-05

5 µmol/~2.2 mg

 特异性Epac激活剂

B 022-25

5×5 µmol

8-pCPT-2'-O-Me-cAMP

cAMP/Epac信号通路研究用试剂

C 041-05

5 µmol/~2.5 mg

 高亲脂性、高膜渗透性的特异性Epac激活剂

C 041-25

5×5 µmol

8-pCPT-2'-O-Me-cAMP-AM

cAMP/Epac信号通路研究用试剂

C 051-01

1 µmol/~0.6 mg

 高膜渗透性特异性Epac激活剂前体

C 051-05

5×1 µmol

Sp-8-pCPT-2'-O-Me-cAMPS

cAMP/Epac信号通路研究用试剂

C 052-01

5 µmol/~2.6 mg

 抗水解性、高亲酯性、高膜渗透性的特异性

 Epac激活剂

Epac阴性对照

产品名称/结构式

产品编号

产品规格

用途

6-Bnz-cAMP

cAMP/Epac信号通路研究用试剂

B 009-10

10 µmol/~4.6 mg

 Epac阴性对照,只激活PKA而不激活Epac

B 009-50,

5×10 µmol

6-Bnz-cAMP-AM

cAMP/Epac信号通路研究用试剂

B 079-01

1 µmol/~0.5 mg

 膜渗透性更佳的Epac阴性对照,

B 079-05

5×1 µmol

Sp-6-Bnz-cAMPS

cAMP/Epac信号通路研究用试剂

B 040-05

5 µmol/~2.4 mg

 抗水解Epac阴性对照

B 040-25

5×5 µmol

8-pCPT-2'-O-Me-cGMP

cAMP/Epac信号通路研究用试剂

C 048-05

5 µmol/~2.6 mg

 基于cGMP结构的Epac阴性对照

C 048-25

5×5 µmol

◆Epac配体

产品名称/结构式

产品编号

产品规格

用途

8-AHA-2'-O-Me-cAMP

cAMP/Epac信号通路研究用试剂

A099-05

5 µmol/~2.3 mg

用于染料或标志物标记的Epac前体

A099-25

5×5 µmol

◆Epac亲和凝胶

产品名称/结构式

产品编号

产品规格

用途

8-AHA-2'-O-Me-cAMP-Agarose

cAMP/Epac信号通路研究用试剂

A057-06

0.6 mL

 封装了Epac激动剂的琼脂糖,适用于cAMP

 相关蛋白的亲和层析。

A057-25

2.5 mL

A057-60

6 mL

荧光Epac激活剂

产品名称/结构式

产品编号

产品规格

特点

8-[DY-547]-AET-2'-O-Me-cAMP

cAMP/Epac信号通路研究用试剂

D089-001

0.1 µmol/~0.1 mg

 荧光Epac激活剂。激发波长= 557 nm,

 发射波长=574 nm。

D089-005

5×0.1 µmol

8-[Pharos-575]-2'-O-Me-cAMP

cAMP/Epac信号通路研究用试剂

P021-001 

0.1 µmol/~74 µg

 膜渗透性荧光Epac激活剂,特别适用于完整

 细胞的研究。

 激发波长= 577 nm,发射波长=605 nm。

P022-005

5×0.1 µmol

※ 本页面产品仅供研究用,研究以外不可使用。

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