Anti-mTOR antibody

5 product images

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  Immunoprecipitation - Anti-mTOR antibody (ab2732)

  mTOR was immunoprecipitated from HeLa cell lysate (1.0 mg per IP reaction; 20% of IP loaded) with ab2732 at 3 μg per reaction. mTOR was also immunoprecipitated by Anti-mTOR antibody ab2833. Western blot was performed from the immunoprecipitate with ab2732 at 1 μg/ml.
  

  Lane 1: Anti-mTOR antibody ab2833 IP in HeLa whole cell lysate.
  Lane 2: ab2732 IP in HeLa whole cell lysate.
  Lane 3: Control IgG IP in HeLa whole cell lysate.

  Detection: Chemiluminescence

  All lanes: Immunoprecipitation - Anti-mTOR antibody (ab2732)

  Predicted band size: 289 kDa

  Exposure time: 3min

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  Western blot - Anti-mTOR antibody (ab2732)

  Lysates prepared using NETN lysis buffer.

  All lanes: Western blot - Anti-mTOR antibody (ab2732) at 0.1 µg/mL

  Lane 1: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 50 µg

  Lane 2: HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 50 µg

  Lane 3: Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 50 µg

  Lane 4: HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 50 µg

  Lane 5: LNCaP (Human prostate cancer cell line) whole cell lysate at 50 µg

  Predicted band size: 289 kDa

  Exposure time: 3min

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  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.
  
  

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  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).

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  Western blot - Anti-mTOR antibody (ab2732)

  False colour image of Western blot: Anti-mTOR antibody staining at 1/2000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab2732 was shown to bind specifically to mTOR. A band was observed at 250 kDa in wild-type A549 cell lysates with no signal observed at this size in MTOR CRISPR-Cas9 edited cell line ab283257. The band observed in the CRISPR-Cas9 edited lysate lane below 250 kDa is likely to represent a truncated form of mTOR. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and MTOR CRISPR-Cas9 edited A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-mTOR antibody (ab2732) at 1/2000 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: MTOR [homo] CRISPR-Cas9 edited A549 cell lysate at 20 µg

  Lane 3: HepG2 cell lysate at 20 µg

  Lane 4: HEK-293 cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

  Lanes 1 - 4: Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 250 kDa