Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-Tuberin antibody [EP1107Y] (ab52936)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP1107Y] to Tuberin
  • Suitable for: WB, IP, Flow Cyt, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Human

Overview

  • Product name

    Anti-Tuberin antibody [EP1107Y]
    See all Tuberin primary antibodies

  • Description

    Rabbit monoclonal [EP1107Y] to Tuberin

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IP, Flow Cyt, IHC-P, ICC/IFmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Synthetic peptide within Human Tuberin aa 1750-1850. The exact sequence is proprietary.

  • Positive control

    • IHC-P: Human adenocarcinoma of uterusIF/ICC: HeLa cell line. WB: SH-SY5Y and HeLa cell lysate Flow Cyt: SH-SY5Y and HeLa cells

  • General notes

    Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.

     

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

Properties

Applications

Our Abpromise guarantee covers the use of ab52936 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 1/20000 - 1/100000. Detects a band of approximately 201 kDa (predicted molecular weight: 201 kDa).
IP 1/70.
Flow Cyt 1/40.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF 1/100.

Target

  • Function

    In complex with TSC1, inhibits the nutrient-mediated or growth factor-stimulated phosphorylation of S6K1 and EIF4EBP1 by negatively regulating mTORC1 signaling. Acts as a GTPase-activating protein (GAP) for the small GTPase RHEB, a direct activator of the protein kinase activity of mTORC1. Implicated as a tumor suppressor. Involved in microtubule-mediated protein transport, but this seems to be due to unregulated mTOR signaling. Stimulates weakly the intrinsic GTPase activity of the Ras-related proteins RAP1A and RAB5 in vitro. Mutations in TSC2 lead to constitutive activation of RAP1A in tumors.

  • Tissue specificity

    Liver, brain, heart, lymphocytes, fibroblasts, biliary epithelium, pancreas, skeletal muscle, kidney, lung and placenta.

  • Involvement in disease

    Defects in TSC2 are the cause of tuberous sclerosis type 2 (TSC2) [MIM:613254]. TSC2 is an autosomal dominant multi-system disorder that affects especially the brain, kidneys, heart, and skin. It is characterized by hamartomas (benign overgrowths predominantly of a cell or tissue type that occurs normally in the organ) and hamartias (developmental abnormalities of tissue combination). Clinical symptoms can range from benign hypopigmented macules of the skin to profound mental retardation with intractable seizures to premature death from a variety of disease-associated causes.
    Defects in TSC2 are a cause of lymphangioleiomyomatosis (LAM) [MIM:606690]. LAM is a progressive and often fatal lung disease characterized by a diffuse proliferation of abnormal smooth muscle cells in the lungs. It affects almost exclusively young women and can occur as an isolated disorder or in association with tuberous sclerosis complex.

  • Sequence similarities

    Contains 1 Rap-GAP domain.

  • Post-translational
    modifications

    Phosphorylation at Ser-1387, Ser-1418 or Ser-1420 does not affect interaction with TSC1.
    Phosphorylation at Ser-939 and Thr-1462 by PKB/AKT1 is induced by growth factor stimulation.

  • Cellular localization

    Cytoplasm. Membrane. At steady state found in association with membranes.
  • Information by UniProt

  • Database links

    • Alternative names

      • FLJ43106 antibody
      • LAM antibody
      • OTTHUMP00000158940 antibody
      • OTTHUMP00000198394 antibody
      • OTTHUMP00000198395 antibody
      • PPP1R160 antibody
      • Protein phosphatase 1, regulatory subunit 160 antibody
      • TSC complex subunit 2 antibody
      • tsc2 antibody
      • TSC2_HUMAN antibody
      • TSC4 antibody
      • TSC4 gene, formerly antibody
      • TSC4, formerly antibody
      • Tuberin antibody
      • Tuberous sclerosis 2 antibody
      • Tuberous sclerosis 2 protein antibody
      • Tuberous sclerosis 2 protein homolog antibody
      see all

    Images

    • Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936)

      Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
      Lane 2: Tuberin knockout HAP1 whole cell lysate (20 µg)
      Lane 3: HeLa whole cell lysate (20 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab52936 observed at 180 kDa. Red - loading control, ab18058, observed at 130 kDa.

      ab52936 was shown to specifically react with tuberin in wild-type HAP1 cells. No band was observed when tuberin knockout samples were used. Wild-type and tuberin knockout samples were subjected to SDS-PAGE.  Ab52936 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Immunocytochemistry/ Immunofluorescence - Anti-Tuberin antibody [EP1107Y] (ab52936)

      ab52936 stained HeLa cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52936 at 1/100 dilution) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

    • Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Anti-Tuberin antibody [EP1107Y] (ab52936) at 1/20000 dilution + HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates prepared in 1%SDS Hot lysis method at 15 µg

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

      Predicted band size: 201 kDa
      Observed band size: 200 kDa
      why is the actual band size different from the predicted?



      Blocking/Diluting Buffer and concentration:
      5% NFDM/TBST

    • Flow Cytometry - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Flow Cytometry - Anti-Tuberin antibody [EP1107Y] (ab52936)

      Overlay histogram showing SH-SY5Y (Human neuroblastoma epithelial cell) cells stained with ab52936 (red line). The cells were fixed with 4% Paraformaldehyde and then permeabilized with 90% Methanol. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) ab150077 at 1/2000 dilution. Isotype control antibody (black line) was rabbit IgG (monoclonal). Unlabelled sample (blue line) was Cell without incubation with primary antibody and secondary antibody.

    • Flow Cytometry - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Flow Cytometry - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Overlay histogram showing HeLa cells stained with ab52936 (red line). The cells were fixed with 80% 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 (ab52936, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
    • Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Western blot - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Anti-Tuberin antibody [EP1107Y] (ab52936) at 1/100000 dilution + SH-SY5Y cell lysate at 10 µg

      Secondary
      HRP-labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 201 kDa
      Observed band size: ~200 kDa why is the actual band size different from the predicted?



      The cell lysates was prepared in 1%SDS Hot lysis method.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tuberin antibody [EP1107Y] (ab52936)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Tuberin antibody [EP1107Y] (ab52936)

      Immunohistochemical analysis of paraffin-embedded human adenocarcinoma of uterus using ab52936 at a dilution of 1/100-1/250.

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    References (3)

    Publishing research using ab52936? Please let us know so that we can cite the reference in this datasheet.

    ab52936 has been referenced in 3 publications.

    • D'Armiento J  et al. Mesenchymal Tumorigenesis Driven by TSC2 Haploinsufficiency Requires HMGA2 and Is Independent of mTOR Pathway Activation. Cancer Res 76:844-54 (2016). PubMed: 26837766
    • Bai Y  et al. Tuberous Sclerosis Complex Protein 2-Independent Activation of mTORC1 by Human Cytomegalovirus pUL38. J Virol 89:7625-35 (2015). Human . PubMed: 25972538
    • Prizant H  et al. Uterine-specific loss of Tsc2 leads to myometrial tumors in both the uterus and lungs. Mol Endocrinol 27:1403-14 (2013). WB ; Mouse . PubMed: 23820898

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