Recombinant
RabMAb

Recombinant Anti-AKT3 (phospho S472) + AKT2 (phospho S474) + AKT1 (phospho S473) antibody [EPR18853] (ab192623)

Overview

  • Product name

    Anti-AKT3 (phospho S472) + AKT2 (phospho S474) + AKT1 (phospho S473) antibody [EPR18853]
  • Description

    Rabbit monoclonal [EPR18853] to AKT3 (phospho S472) + AKT2 (phospho S474) + AKT1 (phospho S473)
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, WB, IP, Dot blotmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human AKT3 aa 450 to the C-terminus (phospho S472). The exact sequence is proprietary.
    Database link: Q9Y243

  • Positive control

    • WB: MCF7 whole cell lysate treated with 100ng/ml IGF-1 for 15 minutes; PC-12 and NIH/3T3 whole cell lysates treated with 100ng/ml PDGF for 60 minutes. ICC/IF: NIH/3T3 cells treated with PDGF (100 ng/ml) for 1 hour. IP: NIH/3T3 treated with 50ng/ml PDGF for 40min whole cell lysate.
  • General notes

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

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab192623 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.
WB 1/1000. Detects a band of approximately 56 kDa (predicted molecular weight: 56 kDa).
IP 1/40.
Dot blot 1/1000.

Target

Images

  • All lanes : Anti-AKT3 (phospho S472) + AKT2 (phospho S474) + AKT1 (phospho S473) antibody [EPR18853] (ab192623) at 1/1000 dilution

    Lane 1 : LNCaP (Human prostate carcinoma epithelial cell) whole cell lysates
    Lane 2 : LNCaP (Human prostate carcinoma epithelial cell) treated with 0.1 uM Calyculin A for 30 minutes whole cell lysates
    Lane 3 : A549 (Human lung carcinoma epithelial cell) whole cell lysates
    Lane 4 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
    Lane 5 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates
    Lane 6 : HUVEC (Human umbilical vein endothelial cell) whole cell lysates
    Lane 7 : C2C12 (Mouse myoblasts myoblast) whole cell lysates
    Lane 8 : Mouse brain lysates
    Lane 9 : Rat heart lysates

    Lysates/proteins at 20 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: 56 kDa


    Exposure time: 50 seconds


    The basal expression level of AKT1 (phospho S473) varies in different cell lines reported by PMID: 19372546. But to detect clear signal, treatment is strongly recommended when using this antibody.

    Blocking and Diluting buffer: 5% NFDM/TBST

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells, untreated or treated with PDGF (100 ng/ml) for 1 hour, labeling AKT3 (phospho S472) + AKT2 (phospho S474) + AKT1 (phospho S473) with ab192623 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

    The signal increased after treatment with PDGF (100 ng/ml) for 1 hour on NIH/3T3 cells.

    The nuclear counter stain is DAPI (blue).

    Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594)) at 1/200 dilution (red).

    Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/1000 dilution.

  • Dot blot analysis of AKT3 (phospho S472) labeled with ab192623 at 1/1000 dilution.

    Lane 1: AKT3 (phospho S472) phospho peptide;

    Lane 2: AKT3 non-phospho peptide;

    Lane 3: AKT1 (phospho S473) phospho peptide;

    Lane 4: AKT2 (phospho S474) phospho peptide.

    Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated (ab97051) at 1/100000 dilution was used as secondary antibody.

    Blocking and diluting buffer: 5% NFDM/TBST.

    Exposure time: 3 minutes.

  • All lanes : Anti-AKT3 (phospho S472) + AKT2 (phospho S474) + AKT1 (phospho S473) antibody [EPR18853] (ab192623) at 1/1000 dilution

    Lane 1 : Untreated MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
    Lane 2 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate treated with 100ng/ml IGF-1 for 15 minutes

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 56 kDa
    Observed band size: 56 kDa


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

    Phosphorylation of AKT at S473 can be induced by IGF-1 treatment according to the literature (PMID: 23638184).

  • All lanes : Anti-AKT3 (phospho S472) + AKT2 (phospho S474) + AKT1 (phospho S473) antibody [EPR18853] (ab192623) at 1/1000 dilution

    Lane 1 : Untreated PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
    Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate treated with 100ng/ml PDGF for 60 minutes
    Lane 3 : Untreated NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
    Lane 4 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate treated with 100ng/ml PDGF for 60 minutes

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 56 kDa
    Observed band size: 56 kDa



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1 and 2: 15 seconds; Lane 3 and 4: 3 minutes.

    Phosphorylation of AKT can be induced by PDGF treatment according to the literature (PMID: 10984605 and 7774014).

  • AKT3 (phospho S472) was immunoprecipitated from 0.35 mg of NIH/3T3 (Mouse embryonic fibroblast cell line) treated with 50ng/ml PDGF for 40min whole cell lysate with ab192623 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab192623 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

    Lane 1: NIH/3T3 treated with 50ng/ml PDGF for 40min whole cell lysate, 10μg (Input).

    Lane 2: ab192623 IP in NIH/3T3 treated with 50ng/ml PDGF for 40min whole cell lysate.

    Lane 3: Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab192623 in NIH/3T3 treated with 50ng/ml PDGF for 40min whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 5 seconds.

References

This product has been referenced in:

  • Li C  et al. SKP2 promotes breast cancer tumorigenesis and radiation tolerance through PDCD4 ubiquitination. J Exp Clin Cancer Res 38:76 (2019). Read more (PubMed: 30760284) »
  • Wang WY  et al. Stimulative role of ST6GALNAC1 in proliferation, migration and invasion of ovarian cancer stem cells via the Akt signaling pathway. Cancer Cell Int 19:86 (2019). Read more (PubMed: 30996686) »
See all 6 Publications for this product

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