Anti-mTOR antibody [Y391]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody [Y391] (AB32028)

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-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-mTOR antibody [Y391] (AB32028)

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.

• IP

Unknown

Immunoprecipitation - Anti-mTOR antibody [Y391] (AB32028)

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.

All lanes:

Immunoprecipitation - Anti-mTOR antibody [Y391] (ab32028)

Predicted band size: 289 kDa

Observed band size: 289 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody [Y391] (AB32028)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody [Y391] (AB32028)

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.

• IP

Unknown

Immunoprecipitation - Anti-mTOR antibody [Y391] (AB32028)

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.

All lanes:

Immunoprecipitation - Anti-mTOR antibody [Y391] (ab32028)

Predicted band size: 289 kDa

Observed band size: 289 kDa

false

• WB

Lab

Western blot - Anti-mTOR antibody [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.

All lanes:

Western blot - Anti-mTOR antibody [Y391] (ab32028) at 1/1000 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

Observed band size: 250 kDa

false

• WB

Lab

Western blot - Anti-mTOR antibody [Y391] (AB32028)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-mTOR antibody [Y391] (ab32028) at 1/2000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates in RIPA buffer at 20 µg

Lane 2:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates in HOT buffer at 20 µg

Lane 3:

Rat brain lysates in RIPA buffer at 20 µg

Lane 4:

Rat brain lysates in HOT buffer at 20 µg

Lane 5:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 6:

Human fetal lung lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 289 kDa

Observed band size: 290 kDa

false

Exposure time: 15s

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-mTOR antibody [Y391] (AB32028)

CUT&RUN profiling with mTOR antibody reveals the expected genomic enrichment pattern in cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with mTOR antibody (Abcam ab32028, 0.5 µg). 500,000 HEK293T cells were used per reaction. IgG, H3K4me3, and H3K27me3 antibodies were included as controls to assess non-specific background, active promoters, and repressed chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Images were generated using Integrative Genomics Viewer (IGV, Broad Institute).

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-mTOR antibody [Y391] (AB32028)

CUT&RUN profiling with mTOR antibody demonstrates robust genome-wide enrichment in cells. Heatmaps of genome-wide signal flanking annotated transcription start sites (TSSs, +/- 2 kbp) display CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with mTOR antibody (Abcam ab32028, 0.5 µg). 500,000 HEK293T cells were used per reaction. IgG antibody was included as a negative control to assess non-specific background. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Heatmaps were generated using ChAsE (Younesy et al., Bioinformatics 2016; PMID 27378294). Row-linked data are ranked by intensity relative to mTOR, with red indicating high localized enrichment and blue denoting background.

• WB

Lab

Western blot - Anti-mTOR antibody [Y391] (AB32028)

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.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-mTOR antibody [Y391] (ab32028) at 1/1000 dilution

Lane 1:

Rat brain tissue lysate at 20 µg

Lane 2:

Rat heart tissue lysate at 20 µg

Lane 3:

Rat liver tissue lysate at 20 µg

Lane 4:

Rat spleen tissue at 20 µg

Lane 5:

Mouse brain tissue lysate at 20 µg

Lane 6:

Mouse heart tissue lysate at 20 µg

Lane 7:

Mouse kidney tissue lysate at 20 µg

Lane 8:

Western blot - Anti-mTOR antibody [Y391] (ab32028) at 1/1000 dilution

Lane 8:

Mouse liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 289 kDa

Observed band size: 289 kDa

false

• WB

Lab

Western blot - Anti-mTOR antibody [Y391] (AB32028)

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.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-mTOR antibody [Y391] (ab32028) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HaCaT (Human skin keratinocyte) whole cell lysate at 20 µg

Lane 3:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Mouse thymus (8-10 weeks) tissue lysate at 20 µg

Lane 5:

Mouse lung (8-10 weeks) tissue lysate at 20 µg

Lane 6:

Rat thymus tissue lysate at 20 µg

Lane 7:

Rat lung tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 289 kDa

Observed band size: 289 kDa

false