MitoSciences (MS1004)

AKT total + Phospho S473 In-Cell ELISA Kit (IR) (ab126579)

Overview

  • Product nameAKT total + Phospho S473 In-Cell ELISA Kit (IR)
  • Detection methodIR
  • 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 ELISA more details
  • PlatformMicroplate

Properties

  • FunctionPlays a role as a key modulator of the AKT-mTOR signaling pathway controlling the tempo of the process of newborn neurons integration during adult neurogenesis, including correct neuron positioning, dendritic development and synapse formation (By similarity). General protein kinase capable of phosphorylating several known proteins. Phosphorylates TBC1D4. Signals downstream of phosphatidylinositol 3-kinase (PI(3)K) to mediate the effects of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). Plays a role in glucose transport by mediating insulin-induced translocation of the GLUT4 glucose transporter to the cell surface. Mediates the antiapoptotic effects of IGF-I. Mediates insulin-stimulated protein synthesis by phosphorylating TSC2 at 'Ser-939' and 'Thr-1462', thereby activating mTORC1 signaling and leading to both phosphorylation of 4E-BP1 and in activation of RPS6KB1. Promotes glycogen synthesis by mediating the insulin-induced activation of glycogen synthase. The activated form can suppress FoxO gene transcription and promote cell cycle progression. Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly.
  • Tissue specificityExpressed in all human cell types so far analyzed. The Tyr-176 phosphorylated form shows a significant increase in expression in breast cancers during the progressive stages i.e. normal to hyperplasia (ADH), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) and lymph node metastatic (LNMM) stages.
  • Involvement in diseaseDefects in AKT1 are a cause of susceptibility to breast cancer (BC) [MIM:114480]. A common malignancy originating from breast epithelial tissue. Breast neoplasms can be distinguished by their histologic pattern. Invasive ductal carcinoma is by far the most common type. Breast cancer is etiologically and genetically heterogeneous. Important genetic factors have been indicated by familial occurrence and bilateral involvement. Mutations at more than one locus can be involved in different families or even in the same case.
    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).
  • 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. The PH domain mediates interaction with TNK2 and Tyr-176 is also essential for this interaction.
    The AGC-kinase C-terminal mediates interaction with THEM4.
  • Post-translational
    modifications
    Phosphorylation 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.
  • Cellular localizationCytoplasm. Nucleus. Cell membrane. Nucleus after activation by integrin-linked protein kinase 1 (ILK1). Nuclear translocation is enhanced by interaction with TCL1A. Phosphorylation on Tyr-176 by TNK2 results in its localization to the cell membrane where it is targeted for further phosphorylations on Thr-308 and Ser-473 leading to its activation and the activated form translocates to the nucleus.
  • Target information above from: UniProt accession P31749 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names
    • AKT1
    • AKT1_HUMAN
    • PKB
    • PKB-ALPHA
    • PRKBA
    • Protein kinase B
    • protein kinase B alpha
    • Proto-oncogene c-Akt
    • RAC
    • rac protein kinase alpha
    • RAC-ALPHA
    • RAC-alpha serine/threonine-protein kinase
    • RAC-PK-alpha
    see all
  • Database links

Applications

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.

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

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.

Protocols

References for AKT total + Phospho S473 In-Cell ELISA Kit (IR) (ab126579)

ab126579 has not yet been referenced specifically in any publications.

Product Wall

We used Janus green as we have experience with this dye in our labs. In addition, we did not use DRAQ5 as this is a registered trade mark from another company and therefore we cannot use it in our kit.
Once the cells are stained with Janus green, ...

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Thank you for your inquiry.
Yes, the 700 channel is the phosphorylated form, the 800 channel is total protein.
Thank you for bringing this to our attention. We are in the process of updating the protocol.

Thank you for your recent telephone enquiry. I appreciate your patience, it has taken some time to confirm this with our Mitosciences laboratories.

They have confirmed that the kit was developed so that AKT1 total could be read in the 800 ch...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"