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Immunofluorescent analysis of phospho-AKT pSer473 (green) in HeLa cells either serum starved overnight (left panel) or treated with 10% FBS (right panel) for 30 minutes. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a phospho-AKT pSer473 monoclonal antibody (Product # OMA1-03061) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:400 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight-554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Synthetic phosphopeptide derived from a region of human PKB that contains serine 473.|
|Storage buffer||Dulbecco's PBS, pH 7.3, with 1mg/ml BSA, 50% glycerol|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Western Blot (WB)||Assay-Dependent|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
OMA1-03061 detects protein kinase B (PKB) phosphorylated at Ser473 from human and mouse samples. OMA1-03061 detects PKB 1,2,and 3.
OMA1-03061 has been successfully used in Western blot, immunofluorescence, and immunocytochemistry procedures. By Western blot, this antibody detects an ~55 kDa protein representing phosphorylated PKB (Ser473) from NIH-3T3 cells treated with platelet-derived growth factor (PDGF).
The OMA1-03061 immunizing phosphopeptide was derived from a region of human PKB that contains serine 473. There is 100% homology with other species, that have not yet been tested.
Antibodies to this protein (and modification) were previously sold as part of a Thermo Scientific Cellomics High Content Screening Kit. This replacement antibody is now recommended for researchers who need an antibody for high content cell based assays. It has been thoroughly tested and validated for cellular immunofluorescence (IF) applications. Further optimization including the selection of the most appropriate fluorescent Dylight conjugated secondary antibody may have to be performed for your high content assay.
Protein kinase B (PKB), also known as Akt, is an oncogenic serine-threonine kinase originally cloned from the retrovirus AKT8. PKB is a direct target of phosphatidylinositol 3-kinase (PI3-kinase) and is activated in response to a variety of growth factors including insulin and platelet-derived growth factor (PDGF). PKB also plays an important role in many PI3-kinase dependent cellular functions including inhibition of apoptosis.
Activity of PKB has been associated with the phosphorylation of two sites: T308, in the activation loop of the kinase, and S473, at the carboxyl terminus. Phosphorylation of both sites contributes to PKB activity, however phosphorylation of T308 has been shown to be absolutely essential for PKB activation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
AKT1 kinase; AKT1m; Akt1m protein; Phospho-AKT; Phospho-PKB; PKB; PKB alpha; Protein kinase B; Protein kinase B alpha; protein kinase B-alpha; Proto-oncogene c-Akt; rac protein kinase alpha; RAC-alpha serine/threonine-protein kinase; RAC-PK-alpha; related to A and C kinases; serine-threonine protein kinase; Thymoma viral proto-oncogene; v-akt murine thymoma viral oncogene homolog 1; v-akt murine thymoma viral oncogene-like protein 1
AKT; AKT1; CWS6; PKB; PKB-ALPHA; PKB/Akt; PKBalpha; PRKBA; RAC; RAC-ALPHA