Anti-mTOR antibody [EPR390(N)]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody [EPR390(N)] (AB134903)

Immunohistochemical analysis of paraffin-embedded A : Wild-type 293T (human embryonic kidney epithelial cell) cell pellet/B : MTOR knockout HEK293T cell pellet labeling mTOR with ab134903 at 1/1000 (2.015 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) wild-type 293T cell pellet, no staining on (B) MTOR knockout HEK293T cell pellet. The section was incubated with ab134903 for 30mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument
Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody [EPR390(N)] (AB134903)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling mTOR with ab134903 at 1/1000 (2.015 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on human colon carcinoma. The section was incubated with ab134903 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument
Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody [EPR390(N)] (AB134903)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling mTOR with ab134903 at 1/1000 (2.015 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on rat colon. The section was incubated with ab134903 for 30mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument
Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mTOR antibody [EPR390(N)] (AB134903)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling mTOR with ab134903 at 1/1000 (2.015 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on mouse colon. The section was incubated with ab134903 for 30mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument
Counterstained with hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Unknown

Western blot - Anti-mTOR antibody [EPR390(N)] (AB134903)

All lanes:

Western blot - Anti-mTOR antibody [EPR390(N)] (ab134903) at 1/10000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

K562 cell lysate at 10 µg

Lane 3:

Raji cell lysate at 10 µg

Lane 4:

HepG2 cell lysate at 10 µg

Lane 5:

293T cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit polyclonal IgG at 1/2000 dilution

Predicted band size: 289 kDa

Observed band size: 289 kDa

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• WB

Lab

Western blot - Anti-mTOR antibody [EPR390(N)] (AB134903)

Blocking buffer and concentration 5% NFDM/TBST
Diluting buffer and concentration 5% NFDM/TBST

Exposure time :
Lane 1 to 5 : 80 seconds
Lane 6 to 13 : 180 seconds

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

All lanes:

Western blot - Anti-mTOR antibody [EPR390(N)] (ab134903) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

Human brain lysates at 20 µg

Lane 4:

Mouse brain lysates at 20 µg

Lane 5:

Rat brain lysates at 20 µg

Lane 6:

Human liver lysates at 20 µg

Lane 7:

Mouse liver lysates at 20 µg

Lane 8:

Human heart lysates at 20 µg

Lane 9:

Mouse heart lysates at 20 µg

Lane 10:

Rat heart lysates at 20 µg

Lane 11:

Human kidney lysates at 20 µg

Lane 12:

Mouse kidney lysates at 20 µg

Lane 13:

Rat kidney 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

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• WB

Lab

Western blot - Anti-mTOR antibody [EPR390(N)] (AB134903)

Lanes 1 - 3 : Merged signal (red and green). Green - ab134903 observed at 289 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab134903 was shown to specifically react with mTOR in wild-type HEK293T cells as signal was lost in MTOR knockout cells. Wild-type and MTOR knockout samples were subjected to SDS-PAGE. ab134903 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-mTOR antibody [EPR390(N)] (ab134903) at 1/10000 dilution

Lane 1:

Wild-type HEK293T whole cell lysate at 20 µg

Lane 2:

MTOR knockout HEK293T whole cell lysate at 20 µg

Lane 3:

K562 whole cell lysate at 20 µg

Predicted band size: 289 kDa

false

• WB

Unknown

Western blot - Anti-mTOR antibody [EPR390(N)] (AB134903)

Lanes 1 - 4 : Merged signal (red and green). Green - ab134903 observed at 289 kDa. Red - loading control, ab130007 observed at 125 kDa.

ab134903 was shown to react with mTOR in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab255411 (knockout cell lysate ab263789) was used. Wild-type and mTOR knockout samples were subjected to SDS-PAGE. ab134903 and Anti-Vinculin antibody [VIN-54] (ab130007) were incubated overnight at 4°C at 1 in 10000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-mTOR antibody [EPR390(N)] (ab134903) at 1/10000 dilution

Lane 1:

Wild-type HEK-293 cell lysate at 20 µg

Lane 2:

MTOR knockout HEK-293 cell lysate at 20 µg

Lane 2:

Western blot - Human MTOR knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-mtor-knockout-hek-293t-cell-line-ab255411'>ab255411</a>)

Lane 3:

Wild-type HEK-293T cell lysate at 20 µg

Lane 4:

MTOR knockout HEK-293T cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 289 kDa

Observed band size: 124 kDa,289 kDa

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