Phospho-AKT (S473) Recombinant Rabbit Monoclonal Antibody [PSH04-44]

☑ Cell treatment (CT)

Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody (HA722129) at 1/2,000 dilution and pan AKT antibody (HA721870) at 1/2,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: MCF7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate
Lane 3: SH-SY5Y cell lysate
Lane 4: SH-SY5Y treated with 100ng/mL PDGF for 5 minutes cell lysate
Lane 5: HEK-293 cell lysate
Lane 6: HEK-293 treated with 50μM LY294002 for 6 hours cell lysate
Lane 7: NIH/3T3 cell lysate
Lane 8: NIH/3T3 treated with 100ng/mL PDGF for 5 minutes cell lysate
Lane 9: C6 cell lysate
Lane 10: C6 treated with 100ng/mL PDGF for 5 minutes cell lysate
Lane 11: MCF7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 20 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% BSA for 1 hour at room temperature. The primary antibody (HA722129) at 1/2,000 dilution and pan AKT antibody (HA721870) at 1/2,000 dilution were used in 5% BSA at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Cell treatment (CT)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue untreated / treated with λpp / phospho-peptide / non-phospho-peptide with Rabbit anti-Phospho-AKT (S473) antibody (HA722129) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722129) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded rat spleen tissue with Rabbit anti-Phospho-AKT (S473) antibody (HA722129) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722129) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-Phospho-AKT (S473) antibody (HA722129) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA722129) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

☑ Cell treatment (CT)

Flow cytometric analysis of NIH/3T3 cells untreated (left) / treated with 100ng/mL PDGF for 1 hour (right) labeling Phospho-AKT (S473).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722129, 0.1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).