Overview

  • Product name

    Anti-Raptor antibody [EP539Y]
    See all Raptor primary antibodies
  • Description

    Rabbit monoclonal [EP539Y] to Raptor
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-Pmore details
    Unsuitable for: Flow Cyt,ICC/IF or IP
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Raptor (C terminal). The exact sequence is proprietary.

  • Positive control

    • Normal human colon and Human cardiac muscle; mouse and rat brain tissue lysate HEK-293, HeLa, MCF-7 and A431 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.

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab40768 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
WB 1/1000 - 1/5000. Detects a band of approximately 140 kDa (predicted molecular weight: 149 kDa).

For unpurified use at 1/500. 

IHC-P 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

  • Application notes
    Is unsuitable for Flow Cyt,ICC/IF or IP.
  • Target

    • Function

      Involved in the control of the mammalian target of rapamycin complex 1 (mTORC1) activity which regulates cell growth and survival, and autophagy in response to nutrient and hormonal signals; functions as a scaffold for recruiting mTORC1 substrates. mTORC1 is activated in response to growth factors or amino-acids. Growth factor-stimulated mTORC1 activation involves a 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-389', which then promotes protein synthesis by phosphorylating PDCD4 and targeting it for degradation.
    • Tissue specificity

      Highly expressed in skeletal muscle, and in a lesser extent in brain, lung, small intestine, kidney and placenta.
    • Sequence similarities

      Belongs to the WD repeat RAPTOR family.
      Contains 7 WD repeats.
    • Cellular localization

      Cytoplasm. Lysosome. Targeting to lysosomes depends on amino acid availability.
    • Information by UniProt
    • Database links

    • Alternative names

      • KIAA1303 antibody
      • KOG1 antibody
      • Mip1 antibody
      • P150 target of rapamycin (TOR) scaffold protein antibody
      • p150 target of rapamycin (TOR) scaffold protein containing WD repeats antibody
      • P150 target of rapamycin (TOR)-scaffold protein antibody
      • Raptor antibody
      • Regulatory associated protein of mTOR antibody
      • Regulatory associated protein of MTOR complex 1 antibody
      • Regulatory-associated protein of mTOR antibody
      • RPTOR antibody
      • RPTOR_HUMAN antibody
      see all

    Images

    • All lanes : Anti-Raptor antibody [EP539Y] (ab40768) at 1/5000 dilution

      Lane 1 : HEK-293 (human embryonic kidney) whole cell lysate
      Lane 2 : HeLa (human cervix adenocarcinoma) whole cell lysate
      Lane 3 : MCF-7 (human breast carcinoma) whole cell lysate

      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: 149 kDa
      Observed band size: 140 kDa
      why is the actual band size different from the predicted?



      Blocking/Diluting buffer 5% NFDM /TBST
    • Immunohistochemical analysis of paraffin-embedded human cardiac muscle sections labelling Raptor with purified ab40768 at dilution of 1/100. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

    • Anti-Raptor antibody [EP539Y] (ab40768) at 1/5000 dilution + Rat brain tissue lysate at 20 mg/ml

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

      Predicted band size: 149 kDa
      Observed band size: 140 kDa why is the actual band size different from the predicted?



      Blocking/Diluting buffer 5% NFDM /TBST
    • Immunohistochemical analysis of paraffin-embedded human colon using anti-Raptor ab40768 at 1/50 dilution.
    • Anti-Raptor antibody [EP539Y] (ab40768) at 1/1000 dilution + Mouse brain tissue lysate at 10 µg

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

      Predicted band size: 149 kDa
      Observed band size: 140 kDa why is the actual band size different from the predicted?



      Blocking/Diluting buffer 5% NFDM /TBST
    • All lanes : Anti-Raptor antibody [EP539Y] (ab40768) at 1/500 dilution

      Lane 1 : 293 cell lysate
      Lane 2 : MCF-7 cell lysate
      Lane 3 : HeLa cell lysate
      Lane 4 : A431 cell lysate

      Lysates/proteins at 10 µg per lane.

      Predicted band size: 149 kDa
      Observed band size: 140 kDa why is the actual band size different from the predicted?
      Additional bands at: 21 kDa, 37 kDa. We are unsure as to the identity of these extra bands.

    References

    This product has been referenced in:

    • Zhang E  et al. Minocycline promotes cardiomyocyte mitochondrial autophagy and cardiomyocyte autophagy to prevent sepsis-induced cardiac dysfunction by Akt/mTOR signaling. Apoptosis 24:369-381 (2019). Read more (PubMed: 30756206) »
    • Vadla R & Haldar D Mammalian target of rapamycin complex 2 (mTORC2) controls glycolytic gene expression by regulating Histone H3 Lysine 56 acetylation. Cell Cycle 17:110-123 (2018). Read more (PubMed: 29143563) »
    See all 14 Publications for this product

    Customer reviews and Q&As

    1-7 of 7 Abreviews or Q&A

    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (Muscle)
    Loading amount
    50 µg
    Specification
    Muscle
    Gel Running Conditions
    Reduced Denaturing
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted May 30 2012

    Question
    Answer

    Thank you very much for taking the time to complete and send the questionnaire.

    The details you have kindly provided will provide us with vital information for our monitoring of product quality.

    Unfortunately none of these antibodies have been tested in frozen sections, and therefore they are not guaranteed in this application, nor do we have specific protocols tips to provide for it.

    However, there are some suggestions that may optimise the antibody performance, if you wish to give them a try:

    -I would recommend decreasing the permeabilization conditions. Both proteins are cytoplasmic; there is no need for such an aggressive permeabilization step. I would repeat the staining skipping this step and removing Triton from the blocking and the washing buffer as well as from the antibody diluent.

    -Use milder blocking conditions, using 5% of serum if possible from the same species as the secondary was raised in, to avoid cross reactivity.

    -Try using more concentrated dilutions, especially for Raptor antibody (1/50 – 1/25).

    If possible I would recommend trying both antibodies in paraffin embedded sections, as they are both guaranteed for it. The link to our protocols page is below, where you can find all the information regarding the different applications protocols, and very useful information for performing paraffin embedded immunostaining.

    https://www.abcam.com/index.html?pageconfig=popular_protocols

    Should the suggestions not improve the results, please do not hesitate to contact me again and I’ll be more than happy to help you further.

    Read More

    Answer

    Thank you for contacting us.

    I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

    In order to better understand the problem, I would like to send you a questionnaire with some additional questions about the protocol used, as well as the order details. All this information is crucial to help us understand the possible causes of the problem. Any images are highly appreciated.

    Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. If no suggestions can be made to the protocol, and the antibody was purchased within the last 6 months, it is covered by our Abpromise guarantee, and therefore I can offer you a free of charge replacement or a credit note for it.

    I look forward to receiving your reply. Please do not hesitate to contact us if you need any more advice or information.

    Read More
    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (Liver)
    Loading amount
    40 µg
    Specification
    Liver
    Gel Running Conditions
    Reduced Denaturing
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Mar 09 2012

    This product is known to not work in this application or species.
    Application
    Immunoprecipitation
    Sample
    Mouse Tissue lysate - whole (Liver)
    Total protein in input
    3000 µg
    Specification
    Liver
    Immuno-precipitation step
    Protein A

    Abcam user community

    Verified customer

    Submitted Mar 09 2012

    Answer

    Thank you for contacting Abcam regarding ab40768. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.  

    Read More

    Answer

    Thank you for contacting us. The secondary antibodies can be used within range from 1/1000 to 1/50,000 dilution. The dilution range depends on the concentration of secondary antibody. We recommend starting with 1/1000, 1/2000, 1/5000 and 1/10,000 for optimization purpose. The dilution factor should also be recommended on vendor's datasheets of secondary antibodies which will give you more idea at what dilution to use. We use BSA in our own lab. You can use milk however if the background will be high then we may recommend switching to BSA. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. 

    Read More

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