WB
Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody at 1/5,000 dilution. Lane 1: MCF7 cell lysate, Lane 2: MCF7 treated with 100nM Calyculin A for 30 minutes cell lysate, Lane 3: NIH/3T3 cell lysate, Lane 4: NIH/3T3 treated with 100ng/mL PDGF for 1 hour cell lysate, Lysates/proteins at 20 µg/Lane. Exposure time: 53 seconds; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.WB
Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody at 1/5,000 dilution. Lane 1: C6 cell lysate Lane 2: C6 treated with 100nM Calyculin A for 30 minutes cell lysate Lysates/proteins at 15 µg/Lane. Exposure time: 3 minutes; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.IHC
Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-Phospho-AKT (S473) antibody 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 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.IHC
Immunohistochemical analysis of paraffin-embedded mouse lung tissue with Rabbit anti-Phospho-AKT (S473) antibody at 1/1,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 at 1/1,000 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.IHC
mmunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-AKT (S473) antibody at 1/1,000 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 at 1/1,000 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.ICC/IF
Immunocytochemistry analysis of HeLa cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-AKT (S473) with Rabbit anti-Phospho-AKT (S473) antibody at 1/200 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-AKT (S473) antibody at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.IF-F
Species: Mouse, Site: Cerebral cortex, Sample: Frozen section, Antibody concentration: 1/200, Antigen retrieval: Not required.IF-P
Species: Human, Site: Breast cancer, Sample: Paraffin-embedded section, Antibody concentration: 1/200.IF-P
Species: Mouse, Site: Lung, Sample: Paraffin-embedded section, Antibody concentration: 1/200.| Product Name | Phospho-AKT (S473) Recombinant Rabbit Monoclonal Antibody |
|---|---|
| Antibody Type | Primary Antibodies |
| Product description | The serine/threonine kinase Akt family contains several members, including Akt1 (also designated PKB or RacPK), Akt2 (also designated PKBβ or RacPK-β) and Akt 3 (also designated PKBγ or thyoma viral proto-oncogene 3), which exhibit sequence homology with the protein kinase A and C families and are encoded by the c-Akt proto-oncogene. All members of the Akt family have a pleckstrin homology domain. Akt1 and Akt2 are activated by PDGF stimulation. This activation is dependent on PDGFR-β tyrosine residues 740 and 751, which bind the subunit of the phosphatidylinositol 3-kinase (PI 3-kinase) complex. Activation of Akt1 by insulin or insulin-growth factor-1(IGF-1) results in phosphorylation of both Thr 308 and Ser 473. Phosphorylation of both residues is important to generate a high level of Akt1 activity, and the phosphorylation of Thr 308 is not dependent on phosphorylation of Ser 473 in vivo. Thus, Akt proteins become phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s). The activation of Akt1 and Akt2 is inhibited by the PI kinase inhibitor wortmannin, suggesting that the protein signals downstream of the PI kinases. |
| Immunogen | Synthetic phospho-peptide corresponding to residues surrounding Ser473 of human AKT1. |
| Modification | p-Ser473 |
| Clonality | Monoclonal |
|---|---|
| Isotype | IgG |
| Host Species | Recombinant rabbit |
| Tested Applications | ICC/IFIF-FIF-PIHCWB |
| WB:1:5000-1:10000 IHC:1:200-1:1000 ICC/IF:1:100-1:1000 IF-F:1:100 IF-P:1:200-1:500 |
|
| Species Reactivity | HumanMouseRat |
| Concentration | 1mg/ml |
| Purification | Protein A |
| Alternative Names | AKT 1 antibody AKT antibody AKT1 antibody AKT1_HUMAN antibody MGC99656 antibody PKB antibody PKB-ALPHA antibody PRKBA antibody Protein Kinase B Alpha antibody Protein kinase B antibody Proto-oncogene c-Akt antibody RAC Alpha antibody RAC antibody RAC-alpha serine/threonine-protein kinase antibody RAC-PK-alpha antibody |
|---|---|
| Molecular Weight(MW) | 56 kDa |
| Cellular Localization | Cytoplasm, Nucleus, Cell membrane. |
WB
Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody at 1/5,000 dilution. Lane 1: MCF7 cell lysate, Lane 2: MCF7 treated with 100nM Calyculin A for 30 minutes cell lysate, Lane 3: NIH/3T3 cell lysate, Lane 4: NIH/3T3 treated with 100ng/mL PDGF for 1 hour cell lysate, Lysates/proteins at 20 µg/Lane. Exposure time: 53 seconds; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
WB
Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody at 1/5,000 dilution. Lane 1: C6 cell lysate Lane 2: C6 treated with 100nM Calyculin A for 30 minutes cell lysate Lysates/proteins at 15 µg/Lane. Exposure time: 3 minutes; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
IHC
Immunohistochemical analysis of paraffin-embedded human breast cancer tissue with Rabbit anti-Phospho-AKT (S473) antibody 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 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.
IHC
Immunohistochemical analysis of paraffin-embedded mouse lung tissue with Rabbit anti-Phospho-AKT (S473) antibody at 1/1,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 at 1/1,000 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.
IHC
mmunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-AKT (S473) antibody at 1/1,000 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 at 1/1,000 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.
ICC/IF
Immunocytochemistry analysis of HeLa cells treated with or without 100nM Calyculin A for 30 minutes labeling Phospho-AKT (S473) with Rabbit anti-Phospho-AKT (S473) antibody at 1/200 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-AKT (S473) antibody at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.
IF-F
Species: Mouse, Site: Cerebral cortex, Sample: Frozen section, Antibody concentration: 1/200, Antigen retrieval: Not required.
IF-P
Species: Human, Site: Breast cancer, Sample: Paraffin-embedded section, Antibody concentration: 1/200.
IF-P
Species: Mouse, Site: Lung, Sample: Paraffin-embedded section, Antibody concentration: 1/200.| Application Notes | WB:1:5000-1:10000 IHC:1:200-1:1000 ICC/IF:1:100-1:1000 IF-F:1:100 IF-P:1:200-1:500 |
|---|
| Form | Liquid |
|---|---|
| Storage Instructions | Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles. |
| Storage Buffer | 1*TBS (pH7.4), 1%BSA, 40%Glycerol. Preservative: 0.05% Sodium Azide. |
