AKT total + Phospho S473 In-Cell ELISA Kit (IR) (ab126579)
- Product nameAKT total + Phospho S473 In-Cell ELISA Kit (IR)
- Tests1 x 96 well plate
- Sample typeAdherent cells, Suspension cells
- Assay typeCell-based
- Species reactivityReacts with: Mouse, Human
- Product overview
Akt is a serine, threonine protein kinase critical in cellular metabolism, glucose uptake, protein synthesis, cell proliferation, growth, apoptosis, survival, angiogenesis, migration and invasion. It acts downstream of the phosphatidylinositol 3 kinase (PI3K) and it mediates the effects of several growth factors such as platelet-derived growth factor, epidermal growth factor and insulin growth factor. It is activated by phosphorylation on Ser-473, Thr-308 and Tyr-474 and when active it phosphorylates transcription factors (FOXO1), kinases (GSK-3, Raf-1, ASK, Chk1) and other signaling proteins (Bad, MDM2). There are three Akt isoforms (Akt1, Akt2 and Akt3) which share 80% sequence identity also known as PKBa, PKBß and PKB?. Akt has been shown to have a role in metabolism, apoptosis and proliferation and therefore it has been proposed to be the candidate “Warburg Kinase”.
- Tested applicationsIn-Cell ELISA more details
- Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 1000X IRDye® Labeled Secondary Antibody Cocktail 1 x 24µl 100X Mouse Anti Akt-1 Primary Antibody 1 x 120µl 100X Rabbit Anti-Akt1 pS473 Primary Antibody 1 x 120µl 100X Triton X-100 1 x 1.25ml 10X Blocking Buffer 1 x 15ml 10X Phosphate Buffered Saline 1 x 100ml 1X Janus Green Stain 1 x 11ml 400X Tween-20 1 x 4ml
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).
Contains 1 AGC-kinase C-terminal domain.
Contains 1 PH domain.
Contains 1 protein kinase domain.
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.
- PKB-ALPHAPRKBAProtein kinase Bprotein kinase B alphaProto-oncogene c-AktRACrac protein kinase alphaRAC-ALPHARAC-alpha serine/threonine-protein kinaseRAC-PK-alpha
Our Abpromise guarantee covers the use of ab126579 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|In-Cell ELISA||In-Cell ELISA|
AKT total + Phospho S473 In-Cell ELISA Kit (IR) images
Figure 1. Normalized IR signal in PDGF induced NIH3T3 cells. NIH3T3 cells were seeded at 40,000 cells per well and allowed to adhere for 2 hours prior to serum starvation and induction of phosphorylation with a dose-response of PDGF recombinant protein. At maximum doses, PDGF induced a 5 fold increase in the levels of Akt-1 phosphorylation.
Figure 2. Normalized IR signal in PDGF induced NIH3T3 cells. NIH3T3 cells were seeded at 40,000 cells per well and allowed to adhere for 2 hours prior to serum starvation and induction of phosphorylation with a dose-response of PDGF recombinant protein. At maximum doses, 1.2 fold induction of Akt-1 is much less than that of the Akt-1 phosphorylation (Figure 1).
Figure 3. Antibody specificity demonstrated by immunocytochemistry. ICC was carried out on PDGF treated NIH3T3 cells with anti-Akt1 phosphoS473 (ab81283) and anti-Akt1 (ab54752) and all buffer reagents as supplied in this kit. Labeling was carried out with a polyclonal antibody GAR-594 and GAM-488 respectively. The PDGF induced cells show significant induction of Akt phosphorylation at residue S473 (observed in the 594 channel).
Figure 4. Antibody specificity demonstrated by immunocytochemistry. ICC was carried out on vehicle treated NIH3T3 cells with anti-Akt1 phosphoS473 (ab81283) and anti-Akt1 (ab54752) and all buffer reagents as supplied in this kit. Labeling was carried out with a polyclonal antibody GAR-594 and GAM-488 respectively. This non-induced control shows less induction than compared to the PDGF induced cells showing a significant induction of Akt phosphorylation at residue S473 (figure 3).
Figure 5. Validation of antibodies by Western Blot. Western blot was run on a 10-20% gradient acrylamide gel. Samples were loaded as follows from left to right: (1) 50ng of Human recombinant AKT1 protein (tagged) (ab62279), (2) 25ug of non-induced NIH3T3 cell extract and (3) 25ug of PDGF induced NIH3T3 cell extract. Membrane Blocking was carried out with 5% Milk+50mM Tris+0.05% Tween-20 pH 7.4, primary antibodies (ab54752 at 5ug/mL left and ab81283 at 1:5000 right) were incubated overnight in 5% BSA+50mM+0.05% Tween-20 pH 7.4 and secondary antibodies were incubated for 2 hours in 5% Milk+50mM Tris+0.05% Tween-20 pH 7.4.
References for AKT total + Phospho S473 In-Cell ELISA Kit (IR) (ab126579)
ab126579 has not yet been referenced specifically in any publications.