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cAMP-dependent Protein Kinase (PKA), catalytic subunit

Catalog # Concentration Size List Price Quantity Your Price
P6000L 2500000 units/ml 500000 units $1,035.00
$931.50
P6000S 2500000 units/ml 100000 units $260.00
$234.00
Catalog # Size List Price Your Price
P6000L 500000 units $1,035.00
$931.50
P6000S 100000 units $260.00
$234.00
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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
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).
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  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
  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 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.

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 EDTA
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


Companion Products
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.
  • This clone has a nucleotide sequence identical to the GenBank entry NM_008854, as entered by Dr. G. S. McKnight.
  • 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
  • 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.
  • Slice, L.W. and Taylor, S.S. (1989). J. Biol. Chem. 264, 20940-20946.
  • Moore, M.J. et al. (2002). J. Biol. Chem. 277, 47878-47884.
  • Zetterqvist, O.Z. et al. (1990). B.E. Kemp, ed(Ed.), in Peptides and Protein Phosphorylation. 171-187. CRC Press, Inc. Boca Raton.
FAQs
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
Quality Control Assay
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety Data Sheets
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