产品信息

• 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