概述

  • 产品名称
    Anti-mTOR抗体[Y391]
    参阅全部 mTOR 一抗
  • 描述
    兔单克隆抗体[Y391] to mTOR
  • 宿主
    Rabbit
  • 经测试应用
    适用于: ChIP, WB, IP, IHC-Pmore details
    不适用于: Flow Cyt or ICC/IF
  • 种属反应性
    与反应: Mouse, Rat, Human
  • 免疫原

    Synthetic peptide within Human mTOR aa 2400-2500 (C terminal). The exact sequence is proprietary.
    (Peptide available as ab193663)

  • 表位
    ab32028 reacts with an epitope located in the C terminal region of mTOR.
  • 阳性对照
    • WB: HeLa, HaCaT and MDA-MB-231 cell lysates and rat brain tissue lysate. IHC-P: Human breast carcinoma, mouse testis and rat testis tissues. IP: Rat brain tissue lysate and HeLa cell lysate.
  • 常规说明

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

    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.

    This product is a recombinant rabbit monoclonal antibody.

性能

应用

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.

应用 Ab评论 说明
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).

This antibody detects non-specific bands and high background. It doesn’t detect the target band in some mouse and rat tissues.

IP 1/50 - 1/100.
IHC-P 1/400. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

  • 应用说明
    Is unsuitable for Flow Cyt or ICC/IF.
  • 靶标

    • 功能
      Kinase 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'.
    • 组织特异性
      Expressed in numerous tissues, with highest levels in testis.
    • 序列相似性
      Belongs to the PI3/PI4-kinase family.
      Contains 1 FAT domain.
      Contains 1 FATC domain.
      Contains 7 HEAT repeats.
      Contains 1 PI3K/PI4K domain.
    • 翻译后修饰
      Autophosphorylated; when part of mTORC1 or mTORC2.
    • 细胞定位
      Endoplasmic 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
    • 数据库链接
    • 别名
      • 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

    图片

    • 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.

    • 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.

    • 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.

    • 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.

    • 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.

    • Lanes 1-7 : Anti-mTOR antibody [Y391] (ab32028)
      Lane 8 : Anti-mTOR antibody [Y391] (ab32028) at 1/1000 dilution

      Lane 1 : Rat brain tissue lysate with 5% NFDM/TBST
      Lane 2 : Rat heart tissue lysate with 5% NFDM/TBST
      Lane 3 : Rat liver tissue lysate with 5% NFDM/TBST
      Lane 4 : Rat spleen tissue with 5% NFDM/TBST
      Lane 5 : Mouse brain tissue lysate with 5% NFDM/TBST
      Lane 6 : Mouse heart tissue lysate with 5% NFDM/TBST
      Lane 7 : Mouse kidney tissue lysate with 5% NFDM/TBST
      Lane 8 : Mouse liver tissue lysate with 5% NFDM /TBST

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

      Predicted band size: 289 kDa
      Observed band size: 289 kDa



      Exposure time:

      Lane 1: 10 seconds
      Lane 2-8: 180 seconds

      This antibody detects non-specific bands and high background. It doesn’t detect the target band in some mouse and rat tissues.

    • All lanes : Anti-mTOR antibody [Y391] (ab32028) at 1/1000 dilution

      Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with 5% NFDM/TBST
      Lane 2 : HaCaT (Human skin keratinocyte) whole cell lysate with 5% NFDM/TBST
      Lane 3 : MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate with 5% NFDM/TBST
      Lane 4 : Mouse thymus (8-10 weeks) tissue lysate with 5% NFDM/TBST
      Lane 5 : Mouse lung (8-10 weeks) tissue lysate with 5% NFDM/TBST
      Lane 6 : Rat thymus tissue lysate with 5% NFDM/TBST
      Lane 7 : Rat lung tissue lysate with 5% NFDM/TBST

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

      Predicted band size: 289 kDa
      Observed band size: 289 kDa



      Exposure time:

      Lane 1-3: 100 seconds
      Lane 4-7: 180 seconds

      This antibody detects non-specific bands and high background. It doesn’t detect the target band in some mouse and rat tissues.

    文献

    This product has been referenced in:
    • Wang P  et al. Resveratrol ameliorates autophagic flux to promote functional recovery in rats after spinal cord injury. Oncotarget 9:8427-8440 (2018). Read more (PubMed: 29492205) »
    • Zhou L  et al. miR-185 Inhibits Fibrogenic Activation of Hepatic Stellate Cells and Prevents Liver Fibrosis. Mol Ther Nucleic Acids 10:91-102 (2018). Read more (PubMed: 29499960) »
    See all 40 Publications for this product

    客户评价及客户问答

    1-9 of 9 Abreviews or Q&A

    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

    Abcam user community

    Verified customer

    提交于 Apr 24 2015

    Answer

    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 purposes only, is *.

    If there is anything else I can help you with, or if you have any issues with the new vial of the antibody, then please let me know.

    Read More

    Answer

    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 only done on Human sample here and not mouse sample. Mouse cross-reactivity was only determined by WB only. Therefore, it could be that, although the antibody is IP'ing the mouse mTOR, the affinity of the antibody to the mouse version may be lower than that to human and that might explain why you are not seeing the co-IP of your other proteins of interest.
    To help try and resolve this situation, I don’t think that any protocol tips would prove to be successful; therefore I would like to suggest that I send ab2732 as a free of charge replacement:

    https://www.abcam.com/mTOR-antibody-ab2732.html



    If you are interested in receiving ab2732, then please let me know, or if there is anything else that I can help you with to resolve this issue. If you could also provide either the Abcam order number or the PO# used to purchase the antibody, that would be very helpful.



    I look forward to your reply.

    Read More

    Answer

    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 issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

    To be able to help you futher, would you be able to send me some information on the protocol that you have been using with ab32028 eg how much antibody you are using, length of incubation, type of samples etc. Also, if possible, it would be very helpful if you could also supply an image of the results that you are getting with the antibody.

    I look forward to your reply and helping to resolve this issue.

    Read More

    Answer

    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 other antibodies, I will let you know.
    I hope this information is of help to you. Please do not hesitate to contact us if you need any more advice or information.
    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    Read More

    Answer

    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, though. A publication used rat samples: Gant JC et al. Disrupting function of FK506-binding protein 1b/12.6 induces the Ca²+-dysregulation aging phenotype in hippocampal neurons. J Neurosci 31:1693-703 (2011). Rat. PubMed: 21289178
    ab51089, ab59299, ab59350: IHC-P was tested, but I have no rat data at hand.
    I will contact the lab to check what rat tissue/samples the antibodies were tested on and let you know.
    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    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

    Abcam user community

    Verified customer

    提交于 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

    Abcam user community

    Verified customer

    提交于 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

    Prof. Paul Townsend

    Verified customer

    提交于 Jun 26 2008

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

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