Anti-TOM70 抗体 [EPR26576-162] (ab289977)

False colour image of Western blot: Anti-TOM70 antibody [EPR26576-162] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab289977 was shown to bind specifically to TOM70. A band was observed at 72 kDa in wild-type HeLa cell lysates with no signal observed at this size in TOMM70 knockout cell line ab265396 (knockout cell lysate ab258732). To generate this image, wild-type and TOMM70 knockout HeLa 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.

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labelling TOM70 with ab289977 at 1/2000 dilution followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human cerebrum. The section was incubated with ab289977 for 10 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 10 mins.

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized C6 (rat glial tumor glial cell) cells labelling TOM70 with ab289977 at 1/500 dilution (0.1µg) (Red)compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized C6 cells labeling TOM70 with ab289977 at 1/100 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in C6 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

TOM70 was immunoprecipitated from C6 (rat glial tumor glial cell) whole cell lysate with ab289977 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab289977 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: C6 (rat glial tumor glial cell) whole cell lysate 10 µg

Lane 2: ab289977 IP in C6 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab289977 in C6 whole cell lysate

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

Exposure time: 3.25 seconds

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

The expression profile/molecular weight observed is consistent with that described in the literature (PMID: 32356556)

Low expression: Breast (human protein atlas: https://www.proteinatlas.org/ENSG00000154174-TOMM70)

Exposure time: 15 seconds

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling TOM70 with ab289977 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labeling TOM70 with ab289977 at 1/100 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluo^r® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized U-2 OS (human bone osteosarcoma epithelial cell) cells labelling TOM70 with ab289977 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labelling TOM70 with ab289977 at 1/2000 dilution followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human breast cancer. The section was incubated with ab289977 for 10 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 10 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-2 OS cells labeling TOM70 with ab289977 at 1/100 dilution, followed by ab150081 Goat Anti-Rbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing mitochondrial staining in U-2 OS cell line. ab33985 Anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used at 1/1000  dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

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

The expression profile/molecular weight observed is consistent with that described in the literature (PMID: 31907385, PMID:11956321).

 Exposure time: 3.25 seconds

Immunohistochemical analysis of paraffin-embedded Human esophagus tissue labelling TOM70 with ab289977 at 1/2000 dilution followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human esophagus. The section was incubated with ab289977 for 10 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 10 mins

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg

Lane 2: ab289977 IP in HeLa whole cell lysate

Lane 3:Rabbit monoclonal IgG (ab172730) instead of ab289977 in HeLa whole cell lysate

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

Exposure time: 3.25 seconds

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling TOM70 with ab289977 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human colon. The section was incubated with ab289977 for 10 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 10 mins

TOM70 was immunoprecipitated from NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab289977 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab289977 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 µg

Lane 2: ab289977 IP in NIH/3T3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab289977 in NIH/3T3 whole cell lysate

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

Exposure time: 3.25 seconds