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MitoSciences (MS1048)

AKT total + Phospho S473 FLOW Kit (ab126580)

Q&A (2)

Overview

  • Product nameAKT total + Phospho S473 FLOW Kit
  • Sample type
    Adherent cells, Suspension cells
  • Assay typeDirect
  • 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 PKBα, PKBβ and PKBγ. 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 applicationsFlow Cyt more details

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.

    • Target information above from: UniProt accession Q9Y243 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
    • Alternative names
        Akt3AKT3_HUMANPKB gamma
        Protein kinase Akt-3Protein kinase B gammaRAC-gamma serine/threonine-protein kinaseRAC-PK-gammaSTK-2
      see all
  • Database links
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    • Applications

    Our Abpromise guarantee covers the use of ab126580 in the following tested applications.

    The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

    Application Notes
    Flow Cyt Flow Cyt

    AKT total + Phospho S473 FLOW Kit images

    • Flow Cytometry - AKT total (s473) FLOW Kit (ab126580)
      Flow Cytometry - AKT total (s473) FLOW Kit (ab126580)
      Figure 1. Antibody specificity demonstrated by Flow cytometry. Non induced (red) and PDGF induced (blue) NIH3T3 were targeted with the antibody cocktail against Akt1. Background fluorescence (black) was determined with a no-primary antibody control. After background subtraction, the PDGF induced cell line shows 1.2 increase in the levels of Akt1 protein.
    • Flow Cytometry - AKT total (s473) FLOW Kit (ab126580)
      Flow Cytometry - AKT total (s473) FLOW Kit (ab126580)
      Figure 2. Antibody specificity demonstrated by Flow cytometry. Non induced (red) and PDGF induced (blue) NIH3T3 were targeted with the antibody cocktail against Akt1 (phospho S473). Background fluorescence (black) was determined with a no-primary antibody control. After background subtraction, the PDGF induced cell line shows a 7 fold increase in the levels of phosphorylated Akt1.
    • Flow Cytometry - AKT total (s473) FLOW Kit (ab126580)
      Flow Cytometry - AKT total (s473) FLOW Kit (ab126580)
      Figure 3. Antibody specificity demonstrated by Flow cytometry. Non induced (vehicle) and PDGF induced NIH3T3 were targeted with the antibody cocktail against Akt1. Background fluorescence was determined with a no-primary antibody control. After background subtraction, the PDGF induced cell line shows 1.2 increase in the levels of Akt1 protein.
    • Flow Cytometry - AKT total (s473) FLOW Kit (ab126580)
      Flow Cytometry - AKT total (s473) FLOW Kit (ab126580)
      Figure 4. Antibody specificity demonstrated by Flow cytometry. Non induced (vehicle) and PDGF induced NIH3T3 were targeted with the antibody cocktail against Akt1 (phospho S473). Background fluorescence was determined with a no-primary antibody control. After background subtraction, the PDGF induced cell line shows a 7 fold increase in the levels of phosphorylated Akt1.
    • ICC AKT Total and pSer473
      ICC AKT Total and pSer473
      Antibody specificity demonstrated by immunocytochemistry. ICC was carried out on NIH3T3 cells treated with PDGF (Left) or vehicle (right) with anti-Akt1 phosphoS473 and anti-Akt1 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 (left) show a significant induction of Akt phosphorylation at residue S473 (observed in the 594 channel) in comparison to the non-induced control (right).
    • Western blot - AKT total (s473) FLOW Kit (ab126580)
      Western blot - AKT total (s473) FLOW Kit (ab126580)
      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 FLOW Kit (ab126580)

    ab126580 has not yet been referenced specifically in any publications.

    Publishing research using ab126580 ? Please let us know so that we can cite the reference in this datasheet

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    It is the catalog # I did started from single cell suspension.

    Thank you

    Thank you for your reply.

    To further investigate this issue, could you please send me the original order details for ab126580, AKT total + Phospho S473 FLOW Kit,as according order history of this product, we have not sold any of these kits yet. ...

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    After step 8.1 of the protocol, the cells do not pellet, but form a sheet on top of the solution that will not disperse or pellet with further centrifugation. Does any special tubes need to be used with this kit?

    Thank you for contacting Abcam.
    I just want to make sure that I wrote the catalogue number down correctly, as we have not sold any ab126580 kits yet.
    I’m not sure why the cells are not pelleting after methanol. I don’t think the tube...

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