RabMAb

Anti-mTOR antibody [Y391] - ChIP Grade (ab32028)

Overview

  • Product nameAnti-mTOR antibody [Y391] - ChIP GradeSee all mTOR primary antibodies ...
  • Description
    Rabbit monoclonal [Y391] to mTOR - ChIP Grade
  • Tested applicationsIHC-P, WB, ICC/IF, Flow Cyt, IP more details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    A synthetic peptide corresponding to residues near the C-term of PI3K/PI4K domain of human mTOR/FRAP was used as an immunogen

  • Epitopeab32028 reacts with an epitope located in the C terminal region of mTOR.
  • Positive control
    • Hela cell lysate, MCF7 cell lysate, human prostate carcinoma.
  • General notes

    Produced under U.S. Patent No. 5,675,063.

    This product is available conjugated to DyLight® 488 see ab139837.

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 Notes
IHC-P Use at an assay dependent dilution.
WB 1/1000 - 1/2000. Detects a band of approximately >250 kDa (predicted molecular weight: 289 kDa).
ICC/IF 1/50 - 1/100.
Flow Cyt 1/50 - 1/80.
IP 1/50.

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.
  • Target information above from: UniProt accession P42345 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    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 protein12-rapamycin associated protein 1 antibody
    • FK506 binding protein12-rapamycin associated protein 2 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 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] - ChIP Grade images

  • Overlay histogram showing HeLa cells stained with ab32028 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32028, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a significantly decreased signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween used under the same conditions.

  • Immunohistochemical analysis of mTOR expression in paraffin-embedded human prostate carcinoma, using 1/250 ab32028.
  • ICC/IF image of ab32028 stained MCF7 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 (ab32028, 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.

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

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


    Predicted band size : 289 kDa
    Observed band size : >250 kDa (why is the actual band size different from the predicted?)

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

This product has been referenced in:
  • Sakamoto K  et al. Peripheral odontogenic keratocyst associated with nevoid basal cell carcinoma syndrome: a case report. Oral Surg Oral Med Oral Pathol Oral Radiol N/A:N/A (2013). IHC ; Human . Read more (PubMed: 24332158) »
  • Fleming GF  et al. Phase II trial of temsirolimus in patients with metastatic breast cancer. Breast Cancer Res Treat 136:355-63 (2012). Read more (PubMed: 22245973) »

See all 7 Publications for this product

Product Wall

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...

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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...

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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...

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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...

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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

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"