MitoSciences (MS1047)

AKT total + Phospho S473 In-Cell ELISA kit (Colorimetric) (ab126578)

Overview

  • Product nameAKT total + Phospho S473 In-Cell ELISA kit (Colorimetric)
  • Detection methodColorimetric
  • Tests
    1 x 96 well plate
  • Sample type
    Adherent cells, Suspension cells
  • Assay typeCell-based (qualitative)
  • Assay durationMultiple steps standard assay
  • Species reactivity
    Reacts 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 ELISAmore details
  • PlatformMicroplate

Properties

  • 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.
  • Post-translational
    modifications
    Phosphorylation 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.
  • Information by UniProt
  • Alternative names
    • Akt3
    • AKT3_HUMAN
    • PKB gamma
    • PKBG
    • PRKBG
    • Protein kinase Akt-3
    • Protein kinase B gamma
    • RAC-gamma
    • RAC-gamma serine/threonine-protein kinase
    • RAC-PK-gamma
    • STK-2
    see all
  • Database links

Applications

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.

Application Abreviews Notes
In-Cell ELISA Use at an assay dependent concentration.

AKT total + Phospho S473 In-Cell ELISA kit (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.

Protocols

References for AKT total + Phospho S473 In-Cell ELISA kit (Colorimetric) (ab126578)

ab126578 has not yet been referenced specifically in any publications.

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The lab let me know that we haven’t tested the mouse monoclonal Akt1 antibody in the kitab126578 with either Akt2 or Akt3. The homology between Akt1 and Akt2 is 82%. Moreover, between Akt1 and Akt3 is 83%. ...

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Thank you for contacting us.

I received the following information from the lab:

The mouse Anti Akt-1 Primary Antibody was raised against recombinant full length protein, corresponding to amino acids 1-481 of Human AKT1.

T...

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