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 148-411. 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 full length protein corresponding to human Akt1, fused to 6X His-Tag at N-terminal, expressed in baculovirus in Sf21 insect. Akt1/PKBa Activation and Phosphorylation Assay: 10ng of recombinant active PDK1 was used to phosphorylate Akt1/PKBa, unactive. The activated Akt1/PKBa in turn phosphorylates the Akt specific substrate peptide, (RPRAATF). The activated Akt1/PKBa can also phosphorylate Crosstide, RPRAATF, GSKa (Ser21), GSK3b (Ser9) and Bad (Ser136). PDK1 Kinase Assay Data: PDK1 was used to phosphorylate unactive Akt1/PKBa in the presence of PIP3 and phospholipids. The activated Akt1/PKBa phosphorylates the Akt specific substrate peptide (RPRAATF). 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.
