AKT total + Phospho S473 In-Cell ELISA (Colorimetric) (ab126578)
- Product nameAKT total + Phospho S473 In-Cell ELISA (Colorimetric)
- Detection methodColorimetric
- Tests1 x 96 well plate
- Sample typeAdherent cells, Suspension cells
- Assay typeCell-based (qualitative)
- Assay durationMultiple steps standard assay
- 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”.
Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.
- Tested applicationsIn-Cell ELISA more details
- Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 100X (Goat anti-mouse) HRP labeled Secondary Antibody 1 x 125µl 100X (Goat anti-rabbit) HRP labeled Secondary Antibody 1 x 125µl 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 HRP Development Solution 1 x 24ml 1X Janus Green Stain 1 x 11ml 400X Tween-20 1 x 4ml
- FunctionIGF-1 leads to the activation of AKT3, which may play a role in regulating cell survival. Capable of phosphorylating several known proteins. Truncated isoform 2/PKB gamma 1 without the second serine phosphorylation site could still be stimulated but to a lesser extent.
- Tissue specificityIn adult tissues, it is highly expressed in brain, lung and kidney, but weakly in heart, testis and liver. In fetal tissues, it is highly expressed in heart, liver and brain and not at all in kidney.
- 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.
modificationsPhosphorylation on Thr-305 and Ser-472 is required for full activity (By similarity). Phosphorylated upon DNA damage, probably by ATM or ATR.
Ubiquitinated. When fully phosphorylated and translocated into the nucleus, undergoes 'Lys-48'-polyubiquitination catalyzed by TTC3, leading to its degradation by the proteasome.
- Cellular localizationCytoplasm. Membrane. Membrane-associated after cell stimulation leading to its translocation.
- PKB gamma
- Protein kinase Akt-3
- Protein kinase B gamma
- RAC-gamma serine/threonine-protein kinase
Our Abpromise guarantee covers the use of ab126578 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 (Colorimetric) 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 (Colorimetric) (ab126578)
ab126578 has not yet been referenced specifically in any publications.