The serine/threonine kinase Akt1 has also been described as Rac-PKa or PKBa. Rac protein kinase family members feature a pleckstrin homology (PH) domain at the amino terminus and a protein-serine/threonine kinase catalytic domain at the carboxy terminus. The PH domain (amino acids 1-106) has been implicated in interactions with other proteins such as G-protein beta gamma subunits, as well as phosphoinositides. The kinase domain is located between residues 148 to 411. These enzymes are activated by diverse ligands such as PGDF, EGF and basic FG in NIH 3T3, Rat-1 or Swiss-3T3 cells. Akt1 is a protein of 480 amino acids and has been observed to show different migratory patterns on a western blot according to the state of phosphorylation of the protein. Phosphatase treatment has been shown to result in inactivation of the protein. Recombinant protein corresponding to full-length human Akt1, containing N-terminal 6X His-Tag, expressed by baculovirus in Sf21 cells. Specific Activity: As Reported Unit Definition: One unit of Akt1/PKBa activity is equal to 1nmol phosphate incorporated into 30uM modified Crosstide (GRPRTSSFAEGKK) per minute at 30C with a final ATP concentration of 100uM. Protein Kinase Assay: 50-500ng of this enzyme phosphorylates the Akt/SGK substrate peptide (RPRAATF). Akt1/PKBa may also be used to phosphorylate GSK3a (Ser21), GSK3b (Ser9), BAD (Ser136), 6-phosphofructo 2-kinase and Crosstide. Immunoprecipitation Kinase Assay: 500ng of the recombinant Akt1/PKBa may be used as a control for IPK activity. Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
