Anti-Akt phospho S473 Monoclonal Antibody

Flow Cytometry results of Anti-AKT pS473 (MOUSE) Monoclonal Antibody. The green histogram represents the A431 cells that were stimulated for 15 minutes with 100 ng/mL EGF. The blue histogram shows the untreated A431 cell population, which is bimodal. Both populations were stained with a 1:50 dilution of the Anti-AKT pS473 (MOUSE) Monoclonal Antibody for 30 mins at 4°C. The secondary antibody, Anti-MOUSE IgG (H&L) (GOAT) Antibody DyLight™ 488 Conjugated was used at a 1:200 dilution for 30 mins at 4°C.

Immunohistochemistry of Mouse anti-AKT pS473 antibody. Tissue: human prostate tissue. Fixation: formalin fixed paraffin embedded. Antigen retrieval: not required. Primary antibody: AKT pS473 antibody at 20 µg/mL for 1 h at RT. Secondary antibody: Dako's Techmate streptavidin-biotin reagents at 1:10,000 for 45 min at RT. Localization: AKT pS473 is nuclear and occasionally cytoplasmic. Staining: AKT pS473 as precipitated red signal with hematoxylin purple nuclear counterstain.

Western Blot of Mouse Anti-AKT pS473 antibody. Lane 1: non-phosphorylated AKT in untreated cells. Lane 2: phosphorylated AKT (indicated by arrowhead at ~56 kDa) on PDGF stimulated NIH/3T3 cell lysates. Load: 10 µg per lane. Primary antibody: AKT pS473 antibody at 1:10,000 in TBS with 0.05% Tween-20 with 1% BSA, for 1 h at 4° C. Secondary antibody: HRP conjugated Gt-a-Mouse IgG was used at a 1:20,000 dilution for 1 h at 4° C with FemtoMax™ enhanced chemiluminescent reagent . Other band(s): none.

Immunofluorescence Microscopy of Mouse Anti-AKTpS473 antibody using STED nanoscopy to evaluate AKT activation and migration. Tissue: A431 cells. Antigen retrieval: Panel A: serum starved,unstimulated cells. Panel B: serum starved, EGF stimulated for 15 mins. A massive increase in AKT-pS473 activation, as measured by intensity signal, peaked at 15 minutes and was associated with depolymerized tubulin. Staining: Panel A shows STED data (AKT-pS473, red channel) collected simultaneously with confocal signal (a-tubulin, green channel). Upon stimulation of cells with EGF, a rapid activation of AKT is observed (Panel B) along with a coincident change in the tubulin organization (yellow signal), as well as an extensive cell shape-change (cell membrane folding) and accumulation of AKTpS473 at the cell periphery.

High resolution STED immunofluorescence nanoscopy of Mouse anti-AKT pS473 antibody. Tissue: A431 cells. The merge images (A) and at high magnification (B) show phosphorylated AKT colocalized with the distal microtubules. Fixation: 4% paraformaldehyde for 5 min and after washes blocked with 10% NGS/0.2% Triton X-100 for 30 min. Antigen retrieval: serum deprivation for 12 h. Primary antibody: AKT pS473 antibody at 10 µg/mL and α-tubulin (cyan) at 1.4 µg/mL for 1 h at RT. Secondary antibody: Atto 647N anti-Mouse IgG (ATTO TEC GmbH), and DyLight™488 anti-Rabbit IgG were used at 1.0 µg/mL for 1h at RT for indirect detection. Localization: AKT pS473 is in the cytoplasm and also organized at the periphery of the cell. Staining: AKT pS473 as red signal with bis-benzimide (blue) nuclear counterstain.

Western Blot of Mouse Anti-Akt pS473 antibody. 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: 10 µg per lane. Primary antibody: rabbit anti-Akt (pan) and mouse anti-Akt pS473 specific antibodies at 1:400 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 Mouse Anti-AKTpS473 antibody. Lane 1: A431 cells. Lane 2: A431 cells stimulated for 15 min with EGF. Load: 35 µg per lane. Primary antibody: AKTpS473 antibody at 1:400 for overnight at 4°C. Secondary antibody: DyLight™649 Conjugated Anti-AKT pS473 Monoclonal Antibody at 1:10,000 for 45 min at RT. Block: Blocking Buffer for Fluorescent Western Blotting overnight at 4°C. Predicted/Observed size: 56kDa. Other band(s): none.

Immunofluorescence confocal microscopy of Mouse Anti-AKT pS473 antibody. Tissue: EGF treated A431 cells. Fixation: 0.5% PFA. Antigen retrieval: EGF 15 min. Primary antibody: AKT pS473 antibody at 10 µg/mL for 1 h at RT. Secondary antibody: DyLight 488™ Goat anti-Rabbit IgG, MAb anti-AKT pS473, atto-647N anti-Mouse IgG (Active Motif). at 1:10,000 for 45 min at RT. Localization: AKT pS473 is nuclear and occasionally cytoplasmic. Staining: AKT pS473 as red signal with tubulin (cyan).

Western Blot of Mouse Anti-Akt pS473 antibody. A: Lane 1) PDGF stimulated NIH 3T3 cells [10µl]. Lane 2) NIH 3T3 cells [10µl]. Lane 3) Hela whole cell lysate [10µl] (weak signal). B: Lane 4) GST negative control protein [100ng]. Lane 5) GST negative control protein [25ng]. Lane 6) AKT 1 recombinant protein [100ng]. Lane 7) AKT 1 recombinant protein [25ng]. Block: 5% BSA overnight at 4°C. Primary antibody: Boster monoclonal anti-AKT antibody (200-301-268S, lot no. 27843) used at 1:1000 for overnight at 4°C. Secondary antibody: HRP Conjugated goat anti-mouse 1:25K for 45 min at RT. Detection: (TMBM-100) for 20 minutes, rinsed with deionized water, dried and scanned on conventional flatbed scanner.