Recombinant
RabMAb

Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)

Overview

  • Product name
    Anti-AKT3 + AKT2 + AKT1 antibody [Y89]
  • Description
    Rabbit monoclonal [Y89] to AKT3 + AKT2 + AKT1
  • Specificity
    This product reacts with AKT1, AKT2 and AKT3.
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-P, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat, Cow
  • Immunogen

    Synthetic peptide within Human AKT1 aa 450 to the C-terminus (C terminal). The exact sequence is proprietary.
    Database link: P31749

  • Positive control
    • MCF7 cell lysate and prostate carcinoma tissue.
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

    A trial size is available to purchase for this antibody.

    Alternative versions available:

    Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (Alexa Fluor® 488) (ab204252)
    Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (Alexa Fluor® 647) (ab204285)

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

Properties

Applications

Our Abpromise guarantee covers the use of ab32505 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
ICC/IF 1/100 - 1/250.
WB 1/2000 - 1/10000. Detects a band of approximately 59 kDa (predicted molecular weight: 56 kDa).
IHC-P 1/100.
Flow Cyt 1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP 1/100.

Target

Images

  • ab32505 staining in SK-N-SH cells treated with alsterpaullone (ab141070), by ICC/IF. Decrease of AKT1 + AKT2 + AKT3 expression correlates with increased concentration of alsterpaullone, as described in literature.
    The cells were incubated at 37°C for 6h in media containing different concentrations of ab141070 (alsterpaullone) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab32505 (1/200 dilution was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • Immunohistochemical analysis of paraffin-embedded prostate carcinoma using ab32505 at 1/100 dilution.
  • All lanes : Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505) at 1/10000 dilution

    Lane 1 : 293T cell lysate transfected with GFP tagged AKT1
    Lane 2 : 293T cell lysate transfected with empty vector
    Lane 3 : 293T cell lysate transfected with GFP tagged AKT3
    Lane 4 : 293T cell lysate transfected with empty vector

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size : 56 kDa
    Observed band size : 82 kDa (why is the actual band size different from the predicted?)


    Exposure time : 8 seconds

    Blocking and diluting buffer and concentration: 5% NFDM/TBST

  • All lanes : Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505) at 1/2000 dilution

    Lane 1 : 293T cell lysate transfected with GFP tagged AKT2
    Lane 2 : 293T cell lysate transfected with empty vector

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size : 56 kDa
    Observed band size : 82 kDa (why is the actual band size different from the predicted?)


    Exposure time : 5 seconds

    Blocking and diluting buffer and concentration: 5% NFDM/TBST

  • Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505) at 1/10000 dilution + MCF-7 cell lysate

    Predicted band size : 56 kDa
    Observed band size : 59 kDa (why is the actual band size different from the predicted?)
  • Overlay histogram showing HeLa cells stained with ab32505 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32505, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

References

This product has been referenced in:
  • Cui Y  et al. In-Depth Proteomic Analysis of the Hippocampus in a Rat Model after Cerebral Ischaemic Injury and Repair by Danhong Injection (DHI). Int J Mol Sci 18:N/A (2017). WB ; Rat . Read more (PubMed: 28672812) »
  • Li Y  et al. MicroRNA-210 induces endothelial cell apoptosis by directly targeting PDK1 in the setting of atherosclerosis. Cell Mol Biol Lett 22:3 (2017). WB . Read more (PubMed: 28536634) »

See all 34 Publications for this product

Customer reviews and Q&As

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Bile duct ligation in Liver)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate buffer ph6 20 min at 97ºC in a PT link
Permeabilization
No
Specification
Bile duct ligation in Liver
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde
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Verified customer

Submitted Sep 24 2015

Application
Western blot
Loading amount
40 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (Cardiomyocytes)
Specification
Cardiomyocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
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Submitted Feb 11 2015

Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing (4–15%)
Sample
Mouse Tissue lysate - whole (Lung)
Specification
Lung
Blocking step
BSA as blocking agent for 15 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

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Verified customer

Submitted Jul 09 2014

Thank you for contacting us.

All the products you shortlisted seem to be suitable for your work, however, looking at the availability of those products, I would recommend the following one that are currently in stock:
Anti-AKT1 (N-term)ant...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Glioblastoma Initiating Cells (Primary cells))
Loading amount
30 µg
Specification
Glioblastoma Initiating Cells (Primary cells)
Gel Running Conditions
Reduced Denaturing (12.5)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C
Username

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Verified customer

Submitted Nov 16 2011

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

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