Product Class: Other

cAMP-dependent Protein Kinase (PKA), catalytic subunit
Gray property icon for NEBU recombinant 30 Gray property icon for heat inactivation 65°

Product Introduction

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
Bioz Badge Exists : True
Catalog # Size Concentration
P6000S 100000 units 2500000 units/ml
P6000L 500000 units 2500000 units/ml

Product Information

Description

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).

Recognition Determinant

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).
This product is related to the following categories:
Protein Kinases
This product can be used in the following applications:
Protein Phosphatases and Kinases

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • NEB # Stored at (°C)
    P6000S -20
    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
  • NEB # Stored at (°C)
    P6000L -20
    Component Name Component # Stored at (°C) Amount Concentration
    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 of enzyme catalyzes the phosphorylation of 1 pmol of LRRASLGQLTQEEE substrate in 20 minutes at 30˚C.

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 EGTA
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

Product Notes

  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.

References

  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.

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

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

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, Safety & Legal

Quality Assurance Statement

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.

Specification Change Notifications

Effective February 25, 2026, the unit definition has been rewritten to reflect a change in the method used to assess the unit. The unit concentration is not affected by this change. Note there have been no changes to the product formulation, the amount of product being supplied, or the specific activity. 

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 DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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