Anti-mTOR antibody (ab2732)

Reviews (13)Q&A (16)References (132)

Overview

  • Product name

    Anti-mTOR antibody
    See all mTOR primary antibodies

  • Description

    Rabbit polyclonal to mTOR

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ICC/IF, WB, IHC-Fr, IP, IHC-Pmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human, Equus
    Predicted to work with: Sheep, Rabbit, Goat, Guinea pig, Cow, Dog, Pig, Ferret, Rhesus monkey, Elephant

  • Immunogen

    Synthetic peptide within Human mTOR aa 200-250. The exact sequence is proprietary.
    Database link: P42345
    (Peptide available as ab39393)

  • Positive control

    • ICC/IF: HepG2 cells. L6 myotubes. IHC-P: Rat and mouse testis tissue. IP: HEK-293T lysates.

Properties

Applications

Our Abpromise guarantee covers the use of ab2732 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 Use at an assay dependent concentration.
WB 1/2000.
IHC-Fr 1/100.
IP 1/1000.
IHC-P 1/2000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

  • Function

    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'.

  • Tissue specificity

    Expressed in numerous tissues, with highest levels in testis.

  • Sequence similarities

    Belongs 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 localization

    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

  • Database links

    see all

  • 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

Images

  • Immunocytochemistry/ Immunofluorescence - Anti-mTOR antibody (ab2732)
    Immunocytochemistry/ Immunofluorescence - Anti-mTOR antibody (ab2732)
    ICC/IF image of ab2732 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2732, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody (ab2732)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody (ab2732)This image is courtesy of an Abreview submitted by Carl Hobbs

    ab2732 staining rat testis sections by IHC-P. The tissue was fixed with formaldehyde and a heat mediated antigen retrival step was performed with citric acid pH 6. Blocking of the sample was done with 1%BSA for 10 minutes at 21°C, followed by staining with ab2732 at 1/2000 in TBS/BSA/azide for 2h at 21°C. A biotinylated goat anti-rabbit polyclonal antibody at 1/200 was used as the secondary antibody.  

    Positive components in the submitted image are indicated by coloured arrowheads: red for densely positive nuclei that appear to be confined to the stem cell layer (immediately above, some spermatogonia exhibit a nuclear speckling). Yellow indicates a moderate cytoplasmic positivity, more evident in some seminiferous tubules (ST). Green for a strong apparently cytoplasmic positivity at the lumenal surface of a proportion of ST.

    See Abreview

  • Immunoprecipitation - Anti-mTOR antibody (ab2732)
    Immunoprecipitation - Anti-mTOR antibody (ab2732)

    Detection of Human mTOR by Western Blot of Immunoprecipitates.
    Samples: Whole cell lysate (0.5 or 1.0 mg per IP reaction; 20% of IP loaded) from 293T cells prepared using lysis buffer.
    Antibodies: Anti-mTOR antibody (ab2732) used for IP at 6 µg per reaction.
    For blotting immunoprecipitated mTOR, ab2732 was used at 0.4 µg/ml.  
    Detection: Chemiluminescence with an exposure time of 3 minutes.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody (ab2732)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody (ab2732)This image is courtesy of an abreview submitted by Carl Hobbs, King's College London, United Kingdom

    ab2732 staining mouse testis sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). The tissue was fixed with formaldehyde and a heat mediated antigen retrival step was performed with citric acid pH 6. Blocking of the sample was done with 1%BSA for 10 minutes at 21°C, followed by staining with ab2732 at 1/3000 in TBS/BSA/azide for 2h at 21°C. A biotinylated goat anti-rabbit polyclonal antibody at 1/200 was used as the secondary antibody.  

    Positive components in the submitted image are indicated by coloured arrowheads: positive nuclei (red) appear to be confined to the stem cell layer of the seminiferous tubules whereas maturing cells appear to have light cytoplasmic positivity. In all tubes there is a lumenal cytoplasmic positivity that varies in intensity (green) between tubules. Leydig cells are also positive (yellow).
    Spaces around the tubes are artefactual.

    See Abreview

  • Immunocytochemistry/ Immunofluorescence - Anti-mTOR antibody (ab2732)
    Immunocytochemistry/ Immunofluorescence - Anti-mTOR antibody (ab2732)
    ab2732 at a 1:100 dilution confocally staining mTOR (red) in L6 myotubes, alongside a nuclear antigen antibody (green).    
  • Immunoprecipitation - Anti-mTOR antibody (ab2732)
    Immunoprecipitation - Anti-mTOR antibody (ab2732)

    mTOR protein kinase assay:

    ab2732 (1:1000 dilution) immunoprecipitates were incubated at 30°C with recombinant 4E-BP1 and 32P-gATP. Autoradiograph shows 32P incorporated into 4E-BP1.

References

This product has been referenced in:

  • Tian Y  et al. Silencing of RHEB inhibits cell proliferation and promotes apoptosis in colorectal cancer cells via inhibition of the mTOR signaling pathway. J Cell Physiol 235:442-453 (2020). Read more (PubMed: 31332784) »
  • Zhu W  et al. Dihydroartemisinin suppresses glycolysis of LNCaP cells by inhibiting PI3K/AKT pathway and downregulating HIF-1a expression. Life Sci 233:116730 (2019). Read more (PubMed: 31390552) »
See all 132 Publications for this product

Publishing research using ab2732? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 29 Abreviews or Q&A

Question

LOT NUMBER GR22858-3 ORDER NUMBER 987281 DESCRIPTION OF THE PROBLEM Non-specific staining in nucleus SAMPLE human cells in culture PRIMARY ANTIBODY Concentration or dilution 1:200 Diluent buffer 1% BSA in PBS Incubation time 2hr Was Buffer PBS Number of washes 3 times DETECTION METHOD Laser scanning confocal microscope (488 argon laser) POSITIVE AND NEGATIVE CONTROLS USED - ANTIBODY STORAGE CONDITIONS 4℃ FIXATION OF SAMPLE If yes: Fixative agent and concentration 4% paraformaldehyde Fixation time 10min Fixation temperature RT (20℃) ANTIGEN RETRIEVAL - PERMEABILIZATION STEP Permeabilizing agent and concentration: incubation in 0.1% triton X-100 PBS for 20 min BLOCKING CONDITIONS Concentration 1% BSA in PBS Blocking time 30min Blocking temperature RT (20℃) Endogenous peroxidases blocked? no SECONDARY ANTIBODY Species: invitrogen A11008 Isotype: IgG Reacts against: rabbit Concentration or dilution 1:1000 Diluent buffer PBS Incubation time 1hr Fluorochrome or enzyme conjugate alexa 488 Was Buffer PBS Number of washes 3 times HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? - ADDITIONAL NOTES -

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Answer

Thank you and your customer for your inquiry.

I believe that the images that your customers provided do show very nice specific staining.

According to the Uniprot homepage (http://www.uniprot.org/uniprot/P42345) mTOR is expressed in the endoplasmic reticulum membrane, the Golgi apparatus membrane, the mitochondrion outer membrane, theLysosome and the Cytoplasm. It shuttles between cytoplasm and nucleus.

If it is in the nucleus, it is located in thePML bodies which is nicely shown by your customers image.

I am happy to confirm that this antibody does not seem to exhibit any un-specific staining and that your customers results are very nice.

I hope I could reassure your customer that their results are specific. Please let me know if your customer has any other questions.

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Western blot abreview for Anti-mTOR antibody

 Good
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - whole cell (PC3 Prostate cancer cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
PC3 Prostate cancer cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Read More

Dr. Dimitra Kalamida

Verified customer

Submitted Jun 12 2019

Flow Cytometry abreview for Anti-mTOR antibody

 Good
Abreviews
abreview image
Application
Flow Cytometry
Sample
Human Cell (DU145 prostate cancer cell line)
Permeabilization
Yes - 0.1% Triton X-100
Gating Strategy
No gating was applied
Specification
DU145 prostate cancer cell line
Preparation
Cell harvesting/tissue preparation method: Cells were trypsinized
Sample buffer: PBS
Fixation
Paraformaldehyde
Read More

Dr. Dimitra Kalamida

Verified customer

Submitted Nov 02 2018

Immunocytochemistry/ Immunofluorescence abreview for Anti-mTOR antibody

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (DU145 Prostate cancer cell line)
Permeabilization
Yes - 0.1% Triton X-100
Specification
DU145 Prostate cancer cell line
Blocking step
BSA as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: RT°C
Fixative
Paraformaldehyde
Read More

Dr. Dimitra Kalamida

Verified customer

Submitted Sep 07 2018

Immunohistochemistry free floating abreview for Anti-mTOR antibody

 Good
Abreviews
abreview image
Application
Immunohistochemistry free floating
Sample
Mouse Tissue sections (kidney)
Specification
kidney
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Abcam user community

Verified customer

Submitted Nov 11 2016

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-mTOR antibody

 Good
Abreviews
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Equus Tissue sections (Lung)
Specification
Lung
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated
Permeabilization
Yes - 0.2% Triton-X100
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 20% · Temperature: 20°C
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Abcam user community

Verified customer

Submitted May 14 2013

Question

could you please let me know which is the volume of AB2732?

Thanks in advance and kind regards

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Answer

Thank you for contacting us.

As we deliver 50 µg at 1mg/ml, the expected volume should be 50ul.

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

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Question

Many thanks for your answer.

Best Regards

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Answer

I am glad you have found the information I provided of help. If I can be of any further assistance, please do not hesitate to let me know.

Until then, I wish you all the best with your research.

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Question

Dear Abcam technical support

I'm interested to buy an antibody against mTOR which works well in Western blot.

According to NCBI the molecular weight of mTOR is 289kDa.
The antibodies against mTOR you are selling i.e. ab2732 show specificity against a protein of 255kDa.

I was wondering where this difference is coming from?

And which of the antibodies against mTOR would you advice me to buy for use in Western blot?

Many thanks

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Answer

Thank you for contacting us and your interest in our products.

When trying to detect a protein of the size of mTOR in Western blotting, there is some movement of the band that would be expected away from the predicted band size. Due to how the ladder and protein runs in the SDS-PAGE gel there can be quite large variations in proteins of this size so the size of 255 kDa observed with ab2732 would not be unexpected. The antibody has been used in the staining of HepG2 cells as well as rat testis, supporting it detecting the expected protein.

I have had a look through the antibodies we have against this target and I would suggest also taking a look at the mouse monoclonal ab87540 which has been used in Western blotting and produced a clean band of ˜268 kDa with HEK293 and human liver tissue (amongst other samples).

However, I'd like to reassure you that both the ab2732 and ab87540 would be covered by the Abpromise guarantee if you intend to use it in Western blotting with human samples. More information on this guarantee can be found from the following link:

https://www.abcam.com/abpromise

I'd also like to draw your attention to our protocol booklet which has some useful advice when performing Western blotting with such large proteins. This booklet can be accessed from the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=13111

I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

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Question

We'll take the credit for now, all the mTOR antibodies you
recommended are variants of the one we've been working with
(ie p mTOR). This is going to be put on hold for us for now.
Thanks for all the help!

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Answer

Thank you for your email.
Your credit note ID is xxx.
I am sorry that this antibody did not perform in rat as stated on the datasheet. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department.
Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.
The credit note ID is for your reference only and does not automatically guarantee the credit.
I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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