Anti-mTOR 抗体 [Y391] (ab32028)

False colour image of Western blot: Anti-mTOR antibody [Y391] staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32028 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.

Blocking and diluting buffer: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling mTOR with purified ab32028 at a dilution of 1/400. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

IHC image of mTOR staining in a section of frozen normal human heart performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab32028, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Exposure time:

Lane 1: 10 seconds
Lane 2-8: 180 seconds

This antibody detects non-specific bands and high background. It doesn’t detect the target band in some mouse and rat tissues.

ab32028 (purified) at a dilution of 1/100 immunoprecipitating mTOR in HeLa whole cell lysate.

Lane 1 (input): HeLa whole cell lysate (10µg)

Lane 2 (+): ab32028 + HeLa whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32028 in HeLa whole cell lysate.

For western blotting, ab131366 VeriBlot for IP Detection Reagent (HRP) was used for detection (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse testis tissue labelling mTOR with purified ab32028 at a dilution of 1/400. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

ab32028 (purified) at a dilution of 1/100 immunoprecipitating mTOR in rat brain tissue lysate.

Lane 1 (input): Rat brain tissue lysate (10µg)

Lane 2 (+): ab32028 + rat brain tissue lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32028 in rat brain tissue lysate.

For western blotting, ab131366 VeriBlot for IP Detection Reagent (HRP) was used for detection (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat testis tissue labelling mTOR with purified ab32028 at a dilution of 1/400. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Exposure time:

Lane 1-3: 100 seconds
Lane 4-7: 180 seconds

This antibody detects non-specific bands and high background. It doesn’t detect the target band in some mouse and rat tissues.