Anti-AKT pS473 (MOUSE) Monoclonal Antibody

Immunohistochemistry - AKT pS473 Monoclonal Antibody

Rockland Immunochemicals' Anti-AKT pS473 monoclonal antibody in immunohistochemistry shows detection of phosphorylated AKT pS473 in human prostate tissue. The antibody was used at 20 µg/mL. The staining is much stronger than the weak basal level of phosphorylation in normal prostate tissue. Tissue was formalin fixed and paraffin embedded. No pre-treatment of sample was required. Signal was developed using Dako's Techmate streptavidin-biotin reagents. Personal communication, Glenna Burmer, Lifespan Biosciences, Seattle, WA.

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Immunofluorescence microscopy - AKT pS473 Monoclonal Antibody

High resolution STED immunofluorescence nanoscopy showing detection of phosphorylated AKT. Rockland Immunochemicals' Anti-AKT pS473 Monoclonal Antibody (p/n 200-301-268) was used at 10 µg/mL for 1 h at RT to stain A431 cells after serum deprivation for 12 h. Cells were fixed in 4% paraformaldehyde for 5 min and after washes blocked with 10% NGS/0.2% Triton X-100 for 30 min. Phosphorylated AKT (red) is observed to be localized in the cytoplasm and also organized at the periphery of the cell. Sequential staining at 1.4 µg/mL for 1 h at RT with rabbit anti-a tubulin (cyan) (p/n 600-401-880) is also shown. The merge images (A) and at high magnification (B) show phosphorylated AKT colocalized with the distal microtubules. High magnification images (D, E and F) show phosphorylated AKT appears to be colocalized to microtubules. Images were taken with a 100x objective on a Leica TCS STED (Stimulated Emission and Depletion), Leica Microsystems, Inc. Exton, PA, USA. 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. Prolong® antifade reagent (Invitrogen) was used to mount slides. Personal Communication, Myriam Gastard, Leica Microsystems Inc, Exton, PA (USA).

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Western blot - AKT pS473 Monoclonal Antibody

Rockland Immunochemicals' Anti-AKT pS473 by western blot shows detection of phosphorylated AKT (indicated by arrowhead at ~56 kDa) on PDGF stimulated NIH/3T3 cell lysates (lane 2). No reactivity is seen for non-phosphorylated AKT in untreated cells (lane 1). Each lane contained approximately 10 µg of lysate. All samples were loaded on to a 4-20% gradient gel for separation. After electrophoresis, the gel was blocked with 5% BLOTTO (p/n B501-0500 in TBS for 90 min at RT. The membrane was probed with the primary antibody at a 1:10,000 dilution in TBS with 0.05% Tween-20 with 1% BSA, for 1 h at 4° C. For detection 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). Images were captured using 2X2 binning for 10-20 sec using a BioSpectrum Imaging System (UVP Ltd.)

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Western blot - Akt pS473 Monoclonal Antibody

Anti-Akt pS473 antibody by fluorescent western blot shows simultaneous detection of unphosphorylated and phosphorylated Akt1 present in serum starved and PDGF stimulated NIH/3T3 whole cell lysates. 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). Both lanes were probed with rabbit anti-Akt (pan) and mouse anti-Akt pS473 specific antibodies. This was followed by detection with DyLight™ 549 conjugated anti-rabbit IgG (green) and DyLight™ 649 conjugated anti-mouse IgG (red) secondary antibodies.

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STED nanoscopy - AKTpS473 Antibody

Anti-AKTpS473 Antibody was used in STED nanoscopy to evaluate AKT activation and migration. A431 cells were serum starved. Epidermal growth factor (EGF) was used to stimulate AKT activation. Panel A shows STED data (AKT-pS473, red channel) collected simultaneously with confocal signal (a-tubulin, green channel) on serum starved but unstimulated cells. Panel B shows STED for the A431 cells stimulated with EGF for 15 min. Serum starved cells (A) show highly organized tubulin, and very little activated AKT (mostly at the cell periphery). 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. A massive increase in AKT-pS473 activation, as measured by intensity signal, peaked at 15 minutes and was associated with depolymerized tubulin.

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Western blot - AKTpS473 Antibody

Anti-AKTpS473 Antibody in Western blot showing detection of AKTpS473 in A431 cells. Epidermal growth factor (EGF) was used to stimulate EGF receptor (EGFR) in A431 cells. Cells were stimulated for 15 min with EGF. The Western blot was blocked with Rockland Immunochemicals’ Blocking Buffer for Fluorescent Western Blotting p/n MB-070, incubated with DyLight™649 Conjugated Anti-AKT pS473 Monoclonal Antibody p/n 200-343-268, and detected using the BioRad VersaDoc MP4000 system.

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Confocal microscopy - AKTpS473 Antibody

Anti-AKTpS473 Antibody used in confocal microscopy shows detection of changes in AKTpS473 localization in EGF treated A431 cells. A Leica TCS SP5 was used to detect tubulin (cyan) stained with Rockland Immunochemicals DyLight 488™ Goat anti-Rabbit IgG, and AKT (red) stained with MAb anti-AKT pS473 and detected with atto-647N anti-Mouse IgG (Active Motif). The images show a weak diffuse staining of AKT in serum starved resting cells ("No treatment"), and a marked activation and migration of AKT to the periphery of the cells upon stimulation with the mitogen EGF ("+ EGF 15 min").

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