PKA Catalytic Subunit, Bovine Heart (cAMP-Dependent Protein Kinase Catalytic Subunit, Protein Kinase A, Catalytic Subunit)

Dry Ice

40000

Supplied as a liquid in 350mM K2HPO4, pH 6.8, 100uM DTT.

Native PKA catalytic subunit purified from bovine heart. The catalytic subunit of PKA dissociates from the PKA holoenzyme following binding of cAMP to the PKA regulatory subunits and phosphorylates target proteins at serine and/or threonine within the recognition motif, Arg-Arg-X-Ser*-X. The purified monomeric catalytic subunit does not require cAMP for activation and is suitable for studies of PKA phosphorylation in vitro with purified substrates or following its introduction into intact cells or cell extracts.

Specific Activity:
≥20, 000units/ug protein

Unit Definition:
One unit is defined as the amount of enzyme required to incorporate 1pmol phosphate into Kemptide substrate per min at 30°C.

Assay Conditions:
The activity of the PKA catalytic subunit is assayed in 40mM Tris-HCl, 20mM magnesium acetate, 200uM [gamma-32P]ATP (500-1000cpm/pmol) with 130uM Kemptide substrate in a total volume of 60ul. The reaction is incubated at 30°C for 5min and terminated by spotting 40ul of the reaction mix onto P-81 filters and washing in 0.5% H3PO4 for 5 min for a total of 4 washes. Following the final wash, the filters are rinsed in ethanol, dried, and counted in the presence of scintillation fluid. A value of 200pmol obtained for Kemptide phosphorylation under these conditions corresponds to 1pmol phosphorylated casein.

Storage and Stability:
Aliquot to avoid repeated freezing and thawing and store at -70°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.