Phospho-AKT (S473) Recombinant Rabbit Monoclonal Antibody [PSH04-44]
cat.: HA722129
| Product Type: | Recombinant Rabbit monoclonal IgG, primary antibodies |
|---|---|
| Species reactivity: | Human, Mouse, Rat |
| Applications: | WB, IHC-P, FC |
| Clonality: | Monoclonal |
| Clone number: | PSH04-44 |
| Form: | Liquid |
| Storage condition: | Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles. |
| Storage buffer: | PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide. |
| Concentration: | 1ug/ul |
| Purification: | Protein A affinity purified. |
| Molecular weight: | Predicted band size: 56 kDa |
| Isotype: | IgG |
| Immunogen: | Synthetic phospho-peptide corresponding to residues surrounding Ser473 of human Akt1. |
| Positive control: | MCF7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate, SH-SY5Y treated with 100ng/mL PDGF for 5 minutes cell lysate, HEK-293 cell lysate, NIH/3T3 treated with 100ng/mL PDGF for 5 minutes cell lysate, C6 treated with 100ng/mL PDGF for 5 minutes cell lysate, mouse spleen tissue, rat spleen tissue, human breast cancer tissue. |
| Subcellular location: | Cytoplasm, Nucleus, Cell membrane. |
| Recommended Dilutions:
WB IHC-P FC |
1:2,000-1:5,000 1:200 1:10,000 |
| Uniprot #: | SwissProt: P31749 Human | P31751 Human | Q9Y243 Human | P31750 Mouse | Q60823 Mouse | Q9WUA6 Mouse | P47196 Rat | P47197 Rat | Q63484 Rat |
| Alternative names: | AKT1 AKT1_HUMAN MGC99656 PKB PKB-ALPHA PRKBA Protein Kinase B Alpha Protein kinase B Proto-oncogene c-Akt RAC Alpha RAC RAC-alpha serine/threonine-protein kinase RAC-PK-alpha akt s473 |
Images
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Fig1:
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. |
|
Fig2:
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) 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. |
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Fig3:
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) 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. |
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Fig4:
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) 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. |
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Fig5:
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). |
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