cAMP-dependent Protein Kinase (PKA), catalytic subunit

Home Protein Tools Products cAMP-dependent Protein Kinase (PKA), catalytic subunit

Catalog #	Concentration	Size	List Price	Your Price
P6000S	2,500,000 units/ml	100,000 units	$179.00	Sign In Sign In
P6000L	2,500,000 units/ml	500,000 units	$714.00	Sign In Sign In

Please enter a quantity for at least one size

Product Information
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 Products

This product can be used in the following applications:

Protein Phosphatases and Kinases
- 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
- Tools & Resources
  - Selection Charts
    
    Protein Kinase Substrate Recognition
- FAQs & Troubleshooting
  - FAQs
    
    How much cAMP-dependent Protein Kinase (NEB# P6000) should be used?
    
    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
- Citations & Technical Literature
  - Citations
    
    Product Citation Tool
    
    Powered by Bioz See more details on Bioz