AKT1 (v-akt Murine Thymoma Viral Oncogene Homolog 1, C-AKT, MGC99656, PKB, PRKBA, Protein kinase B, RAC, RAC-ALPHA, RAC-alpha serine/threonine-protein kinase, RAC-PK-alpha)

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.\n\nThe Akt1 sequence is identical to mouse Akt1 (15/15 amino acids) and strongly conserved in bovine Akt1 (14/15 amino acids) and human Akt1 (13/15 amino acids). Human Akt2 shares 11/15 amino acids with the peptide immunogen sequence. Only a fungal protein shows any significant sequence homology (8/15 amino acids) to the peptide sequence.\n\nImmunoblot Analysis: 0.5-2ug/ml detects Akt in RIPA lysates from human A431 carcinoma\ncells. A431 cell lysate was resolved by electrophoresis, transferred to nitrocellulose and probed with anti-Akt1/PKBa (2ug/ml). Proteins were visualized using a goat antirabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.\n\nIncluded Positive Antigen Control: Non-stimulated A431 lysate. Add 2.5ul of 2-mercaptoethanol/100ul of lysate and boil for 5 minutes to reduce the preparation. Load 20ug of reduced lysate per lane for minigels. \n\nImmunoprecipitation Kinase Assay: 4ug immunoprecipitates active Akt from 1mg of HEK293 cells stimulated with IGF-1. Active kinase was detected by comparing the incorporation of [32P] into Akt/SGK Substrate peptide as catalyzed by immunoprecipitates of lysates made in the presence of 1uM Microcystin.\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. For long-term storage and to avoid repeated freezing and thawing, add sterile glycerol (40-50%), aliquot and store at -20 degrees C or colder. Aliquots are stable for at least 12 months at -20 degrees C. 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.

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