AKT total + Phospho S473 FLOW Kit (ab126580)

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Overview

  • Product name

    AKT total + Phospho S473 FLOW Kit

  • Detection method

    Fluorescent

  • Sample type

    Adherent cells, Suspension cells

  • Assay type

    Direct

  • 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”.

  • Platform

    Flow cytometer

Properties

Associated products

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

This product has been referenced in:

  • Guan Y  et al. Chloride channel-3 is required for efficient tumour cell migration and invasion in human cervical squamous cell carcinoma. Gynecol Oncol 153:661-669 (2019). Read more (PubMed: 30905432) »
  • Zhang H  et al. Inhibition of the PI3K/Akt signaling pathway reverses sorafenib-derived chemo-resistance in hepatocellular carcinoma. Oncol Lett 15:9377-9384 (2018). Read more (PubMed: 29928334) »
See all 2 Publications for this product

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

Customer reviews and Q&As

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1-3 of 3 Abreviews or Q&A

Question

I am working with adherent hepatocyotes and am worried that the protocol calls for using Trypsin for detaching cells as I fear this may cause phosphorylatioon changes. Have you used this kit with hepatocytes and/or do you have information about whether the phosphorylation status is maintained?

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Answer

Thank you for contacting us.
We didn’t test this kit with primary hepatocytes.

In our experiments we didn’t see dephosphorylation of AKT on NIH3T3 cells treated with PDGF and harvested with trypsin. The key is to do a short term trypsin treatment to dislodge the cells from the culture plate and dilute the harvested cells on the media used for treatment. Extensive washes of the media in the absence of phosphatase inhibitors will likely lead to dephosphorylation.

To decrease the time required with trypsin to generate a single cell suspension, we advise to passage the cell line the day before the experiment onto a fresh culture plate.

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Question

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

Thank you

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Answer

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.

Looking forward to your reply and helping you determine what the issues with your cells are.

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Question

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?

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Answer

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 has anything to do with it. Cells should be able to centrifuge in any microfuge tube.
The only thing that occurs to me is that you may not have started with a single cell suspension and what they have is a clump of cells or even more likely the cells are lysed and what they see on top is only debris. During sample preparation for flow cytometry it is extremely important to be very gentle with the cells from harvesting to running through the instrument. No vortexing, no squirting with the pipette and all volumes should be layered gently on top of the cells and cells should be resuspended very gently.
Maybe the flow cytometry guide which is attached would have more tips on sample preparation.
Please let me know if there is anything I can help you with.

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