Immunohistochemistry of A1124-03A. Tissue: (A) normal colon tissue, (B) colon tumor tissue. Fixation: formalin fixed paraffin embedded. Antigen retrieval: not required. Primary antibody: A1124-03A at 1:1,000 dilution for 1h at RT. Secondary antibody: Peroxidase rabbit secondary antibody at 1:10,000 for 45 min at RT. Localization: AKT is nuclear. Staining: AKT as precipitated red signal with hematoxylin purple nuclear counterstain.

Western Blot of A1124-03A. Lane 1: NIH/3T3 whole cell lysate. Load: 20ug per lane. Primary antibody: A1124-03A at 1:500 for overnight at 4°C. Secondary antibody: HRP conjugated Goat anto Rabbit IgG at 1:10,000 preceded color development using Pierce Chemical's SuperSignal™ substrate. Block: MOPS buffer overnight at 4°C. Predicted/Observed size: 56kD, 56kD for AKT. Other band(s): none.

Western Blot of simultaneous detection of unphosphorylated and phosphorylated A1124-03A. Lane 1: unstimulated NIH/3T3 lysates contain inactive unphosphorylated Akt1, green band. Lane 2: PDGF stimulated NIH/3T3 lysate contains both inactive (green band) and activated phosphorylated Akt1 (red band). Load: 35ug per lane. Primary antibody: rabbit anti-Akt (pan) and mouse anti-Akt pS473 specific antibodies at 1:1000 for overnight at 4°C. Secondary antibody: DyLight™ 549 conjugated anti-rabbit IgG (green) and DyLight™ 649 conjugated anti-mouse IgG (red) secondary antibodies at 1:10,000 for 45 min at RT. Block: 5% BLOTTO overnight at 4°C.

Western Blot of A1124-03A. Lane 1: NIR MW protein ladder. Lane 2: AKT1, recombinant. Lane 3: AKT1, phosphatase-treated. Lane 4: AKT1, mutant T308A/S473A. Lane 5: AKT2, recombinant. Lane 6: AKT2, phosphatase-treated. Lane 7: AKT3, recombinant. Lane 8: AKT3, phosphatase-treated. Load: 50ng per lane. Blot A: Anti-Akt pT308 used at 1:2270, Blot B: A1124-03A (1:1000)