New England Biolabs, P6000L, cAMP-dependent Protein Kinase (PKA), catalytic subunit

Related Categories Protein Kinases Applications Protein Phosphatases and Kinases Specification 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, LRRA S LG (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 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 FAQ Q: How much cAMP-dependent Protein Kinase (NEB# P6000) should be used? A: The amount of enzyme required varies when different substrates are used. Start with 1-2 µl for 1µg of protein for one hour in a 10-25 µl reaction. If there is still unphosphorylated material, let the reaction go overnight. Q: What is the consensus sequence for PKA (P6000)? A: The consensus sequence is RRXS/TY, where Y tends to be a hydrophobic residue. A phenylalanine in the nearby sequences tends to be a negative determinant for phosphorylation by PKA