Anti-AKT1 (phospho T308 + S473) antibody (ab66138)
- Product nameAnti-AKT1 (phospho T308 + S473) antibodySee all AKT1 primary antibodies ...
- DescriptionRabbit polyclonal to AKT1 (phospho T308 + S473)
- SpecificityThe motif corresponding to the major autophosphorylation site of the human AKT1 kinase protein. This antibody does not cross react to the non-phosphorylated AKT1 or with other unrelated phosphorylated serine. Ser473 corresponds to QFSYS in human, rat, mouse, chicken and canis of AKT/PKB proteins.
- Tested applicationsIHC-P, ICC/IF, ELISA, Dot Blot, IHC-Fr, WB, IP more details
- Species reactivityReacts with: Mouse, Rat, Chicken, Dog, Human
Synthetic peptide within Human AKT1 (C terminal) (phospho S473). The exact sequence is proprietary.
- Positive control
- Whole cell lysate from PDGF stimulated 3T3 cells. This antibody gave a positive signal in the following Methanol fixed cell lines: MCF-7.
- Storage instructionsStore at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
- Storage bufferPreservative: None
Constituents: Tris buffered saline, pH 7.2, containing antibody stabilizer.
- Concentration information loading...
- PurityProtein A purified
- Purification notesSite-directed adsorption and epitope affinity purification.
- Clonality Polyclonal
- Research Areas
Our Abpromise guarantee covers the use of ab66138 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||IHC-P: Use a concentration of 1 - 2 µg/ml.|
|ICC/IF||ICC/IF: Use a concentration of 1 µg/ml.|
|ELISA||ELISA: Use a concentration of 0.01 - 0.1 µg/ml.|
|Dot Blot||Dot: 1/2000.|
|WB||WB: Use a concentration of 0.1 - 0.2 µg/ml. Predicted molecular weight: 55 kDa.
Blocking is recommended with BSA. Non-fat dry milk and PBS should be avoided.
|IP||IP: Use a concentration of 2 - 5 µg/ml.|
- FunctionPlays a role as a key modulator of the AKT-mTOR signaling pathway controlling the tempo of the process of newborn neurons integration during adult neurogenesis, including correct neuron positioning, dendritic development and synapse formation (By similarity). General protein kinase capable of phosphorylating several known proteins. Phosphorylates TBC1D4. Signals downstream of phosphatidylinositol 3-kinase (PI(3)K) to mediate the effects of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). Plays a role in glucose transport by mediating insulin-induced translocation of the GLUT4 glucose transporter to the cell surface. Mediates the antiapoptotic effects of IGF-I. Mediates insulin-stimulated protein synthesis by phosphorylating TSC2 at 'Ser-939' and 'Thr-1462', thereby activating mTORC1 signaling and leading to both phosphorylation of 4E-BP1 and in activation of RPS6KB1. Promotes glycogen synthesis by mediating the insulin-induced activation of glycogen synthase. The activated form can suppress FoxO gene transcription and promote cell cycle progression. Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly.
- Tissue specificityExpressed in all human cell types so far analyzed. The Tyr-176 phosphorylated form shows a significant increase in expression in breast cancers during the progressive stages i.e. normal to hyperplasia (ADH), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) and lymph node metastatic (LNMM) stages.
- Involvement in diseaseDefects in AKT1 are a cause of susceptibility to breast cancer (BC) [MIM:114480]. A common malignancy originating from breast epithelial tissue. Breast neoplasms can be distinguished by their histologic pattern. Invasive ductal carcinoma is by far the most common type. Breast cancer is etiologically and genetically heterogeneous. Important genetic factors have been indicated by familial occurrence and bilateral involvement. Mutations at more than one locus can be involved in different families or even in the same case.
Defects in AKT1 are associated with colorectal cancer (CRC) [MIM:114500].
Defects in AKT1 are associated with susceptibility to ovarian cancer [MIM:604370]; also called susceptibility to familial breast-ovarian cancer type 1 (BROVCA1).
- Sequence similaritiesBelongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. RAC subfamily.
Contains 1 AGC-kinase C-terminal domain.
Contains 1 PH domain.
Contains 1 protein kinase domain.
- DomainBinding of the PH domain to the phosphatidylinositol 3-kinase alpha (PI(3)K) results in its targeting to the plasma membrane. The PH domain mediates interaction with TNK2 and Tyr-176 is also essential for this interaction.
The AGC-kinase C-terminal mediates interaction with THEM4.
modificationsPhosphorylation on Thr-308, Ser-473 and Tyr-474 is required for full activity. Activated TNK2 phosphorylates it on Tyr-176 resulting in its binding to the anionic plasma membrane phospholipid PA. This phosphorylated form localizes to the cell membrane, where it is targeted by PDPK1 and PDPK2 for further phosphorylations on Thr-308 and Ser-473 leading to its activation. Ser-473 phosphorylation by mTORC2 favors Thr-308 phosphorylation by PDPK1. Ser-473 phosphorylation is enhanced by interaction with AGAP2 isoform 2 (PIKE-A). Ser-473 phosphorylation is enhanced in focal cortical dysplasias with Taylor-type balloon cells.
Ubiquitinated; undergoes both 'Lys-48'- and 'Lys-63'-linked polyubiquitination. TRAF6-induced 'Lys-63'-linked AKT1 ubiquitination is critical for phosphorylation and activation. When ubiquitinated, it translocates to the plasma membrane, where it becomes phosphorylated. When fully phosphorylated and translocated into the nucleus, undergoes 'Lys-48'-polyubiquitination catalyzed by TTC3, leading to its degradation by the proteasome.
- Cellular localizationCytoplasm. Nucleus. Cell membrane. Nucleus after activation by integrin-linked protein kinase 1 (ILK1). Nuclear translocation is enhanced by interaction with TCL1A. Phosphorylation on Tyr-176 by TNK2 results in its localization to the cell membrane where it is targeted for further phosphorylations on Thr-308 and Ser-473 leading to its activation and the activated form translocates to the nucleus.
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Anti-AKT1 (phospho T308 + S473) antibody images
All lanes : Anti-AKT1 (phospho T308 + S473) antibody (ab66138) at 1/1000 dilution
Lane 1 : Whole cell lysate derived from PDGF stimulated 3T3
Lane 2 : Whole cell lysate derived from PDGF stimulated 3T3 with immunising peptide
Lysates/proteins at 10 µg per lane.
Predicted band size : 55 kDa
Observed band size : 60 kDa (why is the actual band size different from the predicted?)
ab66138 staining AKT1 in Mouse embryonic stem cells by Immunocytochemistry/Immunofluorescence. The cells were 4% PFA fixed and permeabilized in 0.1% Triton prior to blocking in 10% serum for 30 minutes at room temperature. The primary antibody was diluted 1/1000 and incubated with the sample for 12 hours at 4°C. The secondary antibody was an Alexa Fluor® 594-conjugated Goat anti-Rabbit polyclonal (ab150160), diluted 1/1500.
ICC/IF image of ab66138 stained MCF-7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab66138 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
ab66138 staining phospho AKT1 in Mouse embryonic tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde, permeabilized with 0.1% Triton and blocked with 10% serum for 1 hour at 22°C. Samples were incubated with primary antibody (1/500 in blocking buffer (1:10)) for 16 hours at 4°C. An Alexa Fluor®488-conjugated Goat anti-rabbit polyclonal (1/1000)ab150077) was used as the secondary antibody.
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 55 kDa
PC12 cells were incubated at 37°C for 30 minutes with vehicle control (0 µM) and different concentrations of cabergoline (ab120564). Increased expression of AKT1 (phospho S473) (ab66138) in PC12 cells correlates with an increase in cabergoline concentration, as described in literature.
Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab66138 at 1/1000 dilution and ab8227 at 1 µg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.
1µg peptide blotted onto a nitrocellulose membrane, followed by ab66138 at 1:2000 dilution. A: AKT1 (pS473) peptide B: AKT1 non-phosphopeptide C: non-related phosphopeptide
References for Anti-AKT1 (phospho T308 + S473) antibody (ab66138)
This product has been referenced in:
- Chase A et al. Ruxolitinib as potential targeted therapy for patients with JAK2 rearrangements. Haematologica 98:404-8 (2013). WB ; Mouse . Read more (PubMed: 22875628) »
- Lim S et al. SNAI1-mediated epithelial-mesenchymal transition confers chemoresistance and cellular plasticity by regulating genes involved in cell death and stem cell maintenance. PLoS One 8:e66558 (2013). WB ; Human . Read more (PubMed: 23799116) »