Overview

  • Product nameAnti-mTOR antibody [Y391]
    See all mTOR primary antibodies
  • Description
    Rabbit monoclonal [Y391] to mTOR
  • Tested applicationsChIP, WB, IP, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human mTOR aa 2400-2500 (C terminal). The exact sequence is proprietary.
    (Peptide available as ab193663)

  • Epitopeab32028 reacts with an epitope located in the C terminal region of mTOR.
  • Positive control
    • WB: HeLa, Jurkat, Raw264.7 and MCF7 cell lysates and rat brain tissue lysate. IHC-P: Human prostate carcinoma, human breast carcinoma, mouse testis and rat testis tissues. IP: Rat brain tissue lysate and HeLa cell lysate.
  • General notes

    This product is a recombinant rabbit monoclonal antibody.

     

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated ‘PUR’ on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

Properties

Applications

Our Abpromise guarantee covers the use of ab32028 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
ChIP Use at an assay dependent concentration. PubMed: 20233713
WB 1/1000 - 1/5000. Detects a band of approximately 250 kDa (predicted molecular weight: 289 kDa).Can be blocked with mTOR peptide (ab193663).
IP 1/50 - 1/100.
IHC-P 1/400. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

  • Application notesIs unsuitable for Flow Cyt or ICC/IF.
  • Target

    • FunctionKinase subunit of both mTORC1 and mTORC2, which regulates cell growth and survival in response to nutrient and hormonal signals. mTORC1 is activated in response to growth factors or amino-acids. Growth factor-stimulated mTORC1 activation involves AKT1-mediated phosphorylation of TSC1-TSC2, which leads to the activation of the RHEB GTPase that potently activates the protein kinase activity of mTORC1. Amino-acid-signaling to mTORC1 requires its relocalization to the lysosomes mediated by the Ragulator complex and the Rag GTPases. Activated mTORC1 up-regulates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. mTORC1 phosphorylates EIF4EBP1 and releases it from inhibiting the elongation initiation factor 4E (eiF4E). mTORC1 phosphorylates and activates S6K1 at 'Thr-421', which then promotes protein synthesis by phosphorylating PDCD4 and targeting it for degradation. Phosphorylates MAF1 leading to attenuation of its RNA polymerase III-repressive function. mTORC2 is also activated by growth. factors, but seems to be nutrient-insensitive. mTORC2 seems to function upstream of Rho GTPases to regulate the actin cytoskeleton, probably by activating one or more Rho-type guanine nucleotide exchange factors. mTORC2 promotes the serum-induced formation of stress-fibers or F-actin. mTORC2 plays a critical role in AKT1 'Ser-473' phosphorylation, which may facilitate the phosphorylation of the activation loop of AKT1 on 'Thr-308' by PDK1 which is a prerequisite for full activation. mTORC2 regulates the phosphorylation of SGK1 at 'Ser-422'. mTORC2 also modulates the phosphorylation of PRKCA on 'Ser-657'.
    • Tissue specificityExpressed in numerous tissues, with highest levels in testis.
    • Sequence similaritiesBelongs to the PI3/PI4-kinase family.
      Contains 1 FAT domain.
      Contains 1 FATC domain.
      Contains 7 HEAT repeats.
      Contains 1 PI3K/PI4K domain.
    • Post-translational
      modifications
      Autophosphorylated; when part of mTORC1 or mTORC2.
    • Cellular localizationEndoplasmic reticulum membrane. Golgi apparatus membrane. Mitochondrion outer membrane. Lysosome. Cytoplasm. Nucleus > PML body. Shuttles between cytoplasm and nucleus. Accumulates in the nucleus in response to hypoxia (By similarity). Targeting to lysosomes depends on amino acid availability and RRAGA and RRAGB.
    • Information by UniProt
    • Database links
    • Alternative names
      • dJ576K7.1 (FK506 binding protein 12 rapamycin associated protein 1) antibody
      • FK506 binding protein 12 rapamycin associated protein 1 antibody
      • FK506 binding protein 12 rapamycin associated protein 2 antibody
      • FK506 binding protein 12 rapamycin complex associated protein 1 antibody
      • FK506-binding protein 12-rapamycin complex-associated protein 1 antibody
      • FKBP rapamycin associated protein antibody
      • FKBP12 rapamycin complex associated protein antibody
      • FKBP12-rapamycin complex-associated protein 1 antibody
      • FKBP12-rapamycin complex-associated protein antibody
      • FLJ44809 antibody
      • FRAP antibody
      • FRAP1 antibody
      • FRAP2 antibody
      • Mammalian target of rapamycin antibody
      • Mechanistic target of rapamycin antibody
      • mTOR antibody
      • MTOR_HUMAN antibody
      • OTTHUMP00000001983 antibody
      • RAFT1 antibody
      • Rapamycin and FKBP12 target 1 antibody
      • Rapamycin associated protein FRAP2 antibody
      • Rapamycin target protein 1 antibody
      • Rapamycin target protein antibody
      • RAPT1 antibody
      • Serine/threonine-protein kinase mTOR antibody
      see all

    Anti-mTOR antibody [Y391] images

    • All lanes : Anti-mTOR antibody [Y391] (ab32028) at 1/5000 dilution (purified)

      Lane 1 : HeLa whole cell lysate
      Lane 2 : Jurkat whole cell lysate
      Lane 3 : Rat brain tissue lysate
      Lane 4 : Raw264.7 whole cell lysate

      Lysates/proteins at 20 µg per lane.

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

      Predicted band size : 289 kDa
      Observed band size : 289 kDa
      Blocking and dilution buffer: 5% NFDM/TBST
    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling mTOR with purified ab32028 at a dilution of 1/400. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse testis tissue labelling mTOR with purified ab32028 at a dilution of 1/400. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat testis tissue labelling mTOR with purified ab32028 at a dilution of 1/400. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • ab32028 (purified) at a dilution of 1/100 immunoprecipitating mTOR in rat brain tissue lysate.

      Lane 1 (input): Rat brain tissue lysate (10µg)

      Lane 2 (+): ab32028 + rat brain tissue lysate.

      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32028 in rat brain tissue lysate.

      For western blotting, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1000).

      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • ab32028 (purified) at a dilution of 1/100 immunoprecipitating mTOR in HeLa whole cell lysate.

      Lane 1 (input): HeLa whole cell lysate (10µg)

      Lane 2 (+): ab32028 + HeLa whole cell lysate.

      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32028 in HeLa whole cell lysate.

      For western blotting, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1000).

      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • All lanes : Anti-mTOR antibody [Y391] (ab32028) at 1/2000 dilution (unpurified)

      Lane 1 : HeLa cell lysate
      Lane 2 : MCF-7 cell lysate


      Predicted band size : 289 kDa
      Observed band size : >250 kDa (why is the actual band size different from the predicted?)
    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human prostate carcinoma labelling mTOR with unpurified ab32028 at a dilution of 1/250.

    References for Anti-mTOR antibody [Y391] (ab32028)

    This product has been referenced in:
    • Atif F  et al. Anti-tumor effects of progesterone in human glioblastoma multiforme: role of PI3K/Akt/mTOR signaling. J Steroid Biochem Mol Biol 146:62-73 (2015). Read more (PubMed: 24787660) »
    • Atif F  et al. The Synergistic Effect of Combination Progesterone and Temozolomide on Human Glioblastoma Cells. PLoS One 10:e0131441 (2015). WB ; Human . Read more (PubMed: 26110872) »

    See all 10 Publications for this product

    Product Wall

    Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample Human Tissue sections (prostate cancer)
    Antigen retrieval step Heat mediated - Buffer/Enzyme Used: EDTA, pH9, 100C 20 min
    Permeabilization No
    Specification prostate cancer
    Blocking step H2O2 as blocking agent for 10 minute(s) · Concentration: 3% · Temperature: 25°C
    Fixative Formaldehyde
    Username

    Abcam user community

    Verified customer

    Submitted Apr 24 2015

    Thank you for your reply.

    As requested, I am sending you a new vial of mTOR antibody, ab2732 to use in your IP experiments, since the original vial of antibody was not working as well as it should have. Your new order number, for tracking purp...

    Read More

    Thank you for your patience whilst I was looking in to this matter.

    I checked the concentration for this lot and it is around 1.758 mg/ml. So, I don't believe it is a concentration issue.

    However, I did notice that IP testing was onl...

    Read More

    Thank you for contacting Abcam.

    I am sorry that you are having issues with ab32028 in IP. The antibody is covered under our Abpromise for six months and is guaranteed to work in IP on mouse, rat and human samples . If we cannot resolve the iss...

    Read More

    Thank you for your email.
    I have heard back regarding ab32028:
    The lab tested WB on C6 and PC-12 cell lysates as well as on rat brain tissue lysates. They did not test rat tissues in IHC-P.
    As soon as I have more information for the othe...

    Read More

    Thank you for your reply.
    The following mTOR antibodies have been tested in rat as well as IHC-P:
    ab32028, ab51089, ab59299, and ab59350.
    The antibody ab32028 has been tested in the lab on IHC-P. I am not certain that rat tissue was used...

    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 (HUVEC)
    Loading amount 45 µg
    Specification HUVEC
    Treatment FGF2 50 ng/ml for 5 minutes
    Gel Running Conditions Reduced Denaturing (gradient 4-15%)
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Username

    Abcam user community

    Verified customer

    Submitted Sep 17 2008

    Application Western blot
    Sample Mouse Tissue lysate - whole (ventricular tissue lysates)
    Loading amount 100 µg
    Specification ventricular tissue lysates
    Gel Running Conditions Reduced Denaturing (4 ~ 12% gradient NuPAGE)
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C
    Username

    Abcam user community

    Verified customer

    Submitted Jul 05 2008

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Sample Human Cell lysate - whole cell (Keratinocyte)
    Loading amount 15 µg
    Specification Keratinocyte
    Gel Running Conditions Reduced Denaturing (10% gel)
    Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
    Username

    Prof. Paul Townsend

    Verified customer

    Submitted Jun 26 2008

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