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Anti-AKT1 Antibody 17F6.B11
Also for AKT1 (NM_001014431)
|Anti-AKT pS473 (MOUSE) Monoclonal Antibody was produced by repeated immunizations with a synthetic peptide corresponding to residues surrounding S473 of human AKT1 protein, followed by hybridoma development.|
|human, mouse, rat, monkey
|WB, IHC, IF
||ELISA: 1:20,000, WB: 1:500-1:3,000, IHC: 20 ug/mL, IF: 1:500-1:3,000
|0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2|
|Phospho AKT antibody is suitable for ELISA, immunohistochemistry, immunoprecipitation and western blotting. AKT phospho 473 is a component of the PI-3 kinase pathway and is activated by phosphorylation at Ser 473 and Thr 308. AKT is a cytoplasmic protein also known as AKT1, Protein Kinase B (PKB) and rac (related to A and C kinases). AKT is a key regulator of many signal transduction pathways. AKT Exhibits tight control over cell proliferation and cell viability. Overexpression or inappropriate activation of AKT is noted in many types of cancer. AKT mediates many of the downstream events of PI 3-kinase (a lipid kinase activated by growth factors, cytokines and insulin). PI 3-kinase recruits AKT to the membrane, where it is activated by PDK1 phosphorylation. Once phosphorylated, AKT dissociates from the membrane and phosphorylates targets in the cytoplasm and the cell nucleus. AKT has two main roles: (i) inhibition of apoptosis; (ii) promotion of proliferation. Anti-AKT pS473 (MOUSE) Monoclonal Antibody is ideal for investigators involved in Cell Signaling, Cancer, Neuroscience, Signal Transduction research.
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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 (p/n 610-103-121) was used at a 1:20,000 dilution for 1 h at 4° C with FemtoMax™ enhanced chemiluminescent reagent (p/n FEMTOMAX-100). Other band(s): none.
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 p/n 200-343-268 at 1:10,000 for 45 min at RT. Block: Blocking Buffer for Fluorescent Western Blotting p/n MB-070 overnight at 4°C. Predicted/Observed size: 56kDa. Other band(s): none.
Western Blot of Mouse Anti-Akt pS473 antibody. A Lane 1) PDGF stimulated NIH 3T3 cells 10 Âµl Lane 2) NIH 3T3 cells 10 Âµl Lane 3) Hela whole cell lysate 10 Âµl (weak signal) B Lane 4) GST negative control protein 100 ng Lane 5) GST negative control protein 25 ng Lane 6) AKT 1 recombinant protein 100 ng Lane 7) AKT 1 recombinant protein 25 ng Block: 5% BSA overnight at 4Â°C. Primary antibody: monoclonal anti AKT antibody 200-301-268S, lot no. 27843 used at 1:1000 for overnight at 4Â°C. Secondary antibody: HRP Conjugated goat anti mouse lot 20121 1:25K for 45 min at RT. Detection : TMBM-100 for 20 minutes, rinsed with deionized water, dried and scanned on conventional flatbed scanner
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.
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 a-tubulin (cyan) (p/n 600-401-880) 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 (p/n 611-141-122) 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.
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).