Recombinant
RabMAb

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

Reviews (6)Q&A (1)References (97)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [Y89] to AKT3 + AKT2 + AKT1
  • Suitable for: ICC/IF, WB, IHC-P, Flow Cyt, IP
  • Reacts with: Human

Overview

  • Product name

    Anti-AKT3 + AKT2 + AKT1 antibody [Y89]

  • Description

    Rabbit monoclonal [Y89] to AKT3 + AKT2 + AKT1

  • Host species

    Rabbit

  • 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: Human
    Predicted to work with: Mouse, 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. IP: MCF7 cell lysate

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    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. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt 1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP 1/50.

Target

Images

  • Western blot - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    Western blot - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    All lanes : Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505) at 1/10000 dilution

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

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : 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

  • Immunocytochemistry/ Immunofluorescence - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    Immunocytochemistry/ Immunofluorescence - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)

    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.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)

    Immunohistochemical analysis of paraffin-embedded prostate carcinoma using ab32505 at 1/100 dilution.

    Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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

    Purified ab32505 at 1/50 dilution (2µg) immunoprecipitating AKT3+AKT2+AKT1 in MCF7 whole cell lysate.
    Lane 1 (input): MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10µg
    Lane 2 (+): ab32505 + MCF7 whole cell lysate.
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32505 in MCF7 whole cell lysate.
    VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
    Blocking Buffer and concentration: 5% NFDM/TBST.
    Diluting buffer and concentration: 5% NFDM/TBST.
    Observed band size: 59 kDa

  • Western blot - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    Western blot - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    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
    All lanes : 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

  • Western blot - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    Western blot - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    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?

  • Flow Cytometry - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    Flow Cytometry - Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505)
    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.

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (97)

Publishing research using ab32505? Please let us know so that we can cite the reference in this datasheet.

ab32505 has been referenced in 97 publications.

  • Liu Y  et al. Oxymatrine protects neonatal rat against hypoxic-ischemic brain damage via PI3K/Akt/GSK3ß pathway. Life Sci 254:116444 (2020). PubMed: 31102745
  • Yang HZ  et al. Resveratrol inhibits the proliferation of melanoma cells by modulating cell cycle. Int J Food Sci Nutr 71:84-93 (2020). PubMed: 31154861
  • Yao J  et al. lncRNA GAS5/miR-223/NAMPT axis modulates the cell proliferation and senescence of endothelial progenitor cells through PI3K/AKT signaling. J Cell Biochem N/A:N/A (2019). PubMed: 31026096
  • Xing F  et al. Wogonin Increases Cisplatin Sensitivity in Ovarian Cancer Cells Through Inhibition of the Phosphatidylinositol 3-Kinase (PI3K)/Akt Pathway. Med Sci Monit 25:6007-6014 (2019). PubMed: 31402794
  • Jin J  et al. Cardamonin inhibits breast cancer growth by repressing HIF-1a-dependent metabolic reprogramming. J Exp Clin Cancer Res 38:377 (2019). PubMed: 31455352
View all Publications for this product

Customer reviews and Q&As

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1-7 of 7 Abreviews or Q&A

Immunocytochemistry abreview for Anti-AKT3 + AKT2 + AKT1 antibody [Y89]

 Good
Abreviews
abreview image
Application
Immunocytochemistry
Sample
Rat Cultured Cells (Cardiomyocytes)
Permeabilization
Yes - Triton x-100, 0.03%
Specification
Cardiomyocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde
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Abcam user community

Verified customer

Submitted Feb 08 2018

Immunocytochemistry abreview for Anti-AKT3 + AKT2 + AKT1 antibody [Y89]

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry
Sample
Mouse Cultured Cells (Cardiomyocytes)
Permeabilization
Yes - Triton x-100, 0.03%
Specification
Cardiomyocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Jan 31 2018

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-AKT1 antibody [Y89]

 Good
Abreviews
abreview image
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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Abcam user community

Verified customer

Submitted Sep 24 2015

Western blot abreview for Anti-AKT1 antibody [Y89]

 Excellent
Abreviews
abreview image
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%
Read More

Abcam user community

Verified customer

Submitted Feb 11 2015

Western blot abreview for Anti-AKT1 [Y89] antibody

 Excellent
Abreviews
abreview image
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
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Abcam user community

Verified customer

Submitted Jul 09 2014

Question

Hi,

I was hoping for some advice regarding your antibodies against both AKT 1 or AKT 2 (separately not pan-AKT). I am interested in blotting for truncated mutants of both and therefore need Abs that recognise sequence as near as possible to the N and C terminals. I have narrowed it down to a couple for each situation and, based on more specific details of the immunogen that you may have compared to what is detailed on your website, I wondered if you could recommend which might be best for my purposes (ie. which will bind closest to either the N or C terminal).

AKT 1



N-term; Either ab25893 “Synthetic peptide, consisting of 16 N terminal amino acids of Human AKT1” or ab64936 “Synthetic peptide (Human) from the N terminal region.”



C-term; Either ab32505 “synthetic peptide corresponding to residues in C-terminus of human Akt1.” or ab6076 “Synthetic peptide mapping to the carboxy terminal domain of human AKT1.”



AKT 2



N-term; ab130255 “Recombinant fragment, corresponding to amino acids 2-149 of Human AKT2”, seems to be the best option.



C-term; Either ab39663 “against synthesized non phosphopeptide derived from human AKT2 around the phosphorylation site of serine 474.” or ab5920 “Synthetic peptide (Human)(C terminal).”



Thanks in advance for your help,

Read More
Answer

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)antibody (ab64936), https://www.abcam.com/ab64693
Anti-AKT1 (C-term) antibody [Y89] (ab32505), https://www.abcam.com/ab32505
Anti-AKT2 (phospho S474) antibody (ab39663), https://www.abcam.com/ab39663

At Abcam, providing quality service is just as important as providing quality products. Our Abpromise® ensures that you can trust our products to perform as stated on the datasheets, or we will offer a replacement, credit, or refund.

The Abcam Abpromise® guarantee:
- 100% Scientific and Customer Support for any product you buy from Abcam or one of our authorized distributors.
- We guarantee our products work in the tested species and applications stated on the datasheet.
- We will replace or refund products not performing as stated on the datasheet if reported within 6 months of purchase.
- We investigate any quality concerns raised by customers to ensure our catalog contains products that perform to the highest standards.

Please note these conditions to our Abpromise®:
- Protocol information must be provided in order for the claim to be validated.
- Antibodies tested in recombinant samples only are not guaranteed for use on endogenous samples.
- “Predicted to react” information on the datasheets is provided for reference only and these species are not guaranteed.
- Fast Track antibodies are covered for ELISA against the immunizing peptide only.

I hope this information is helpful, but please do not hesitate to contact us with further questions.


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Western blot abreview for Anti-AKT1 antibody [Y89]

 Excellent
Abreviews
abreview image
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
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Abcam user community

Verified customer

Submitted Nov 16 2011

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