Anti-ATG7 antibody [EPR6251]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ATG7 antibody [EPR6251] (AB133528)

Immunocytochemistry/Immunofluorescence analysis of HT-29 (Human colorectal adenocarcinoma cell line) labeling ATG7 with purified ab133528 at 1/500 dilution. Cells were fixed with 100% methanol. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ATG7 antibody [EPR6251] (AB133528)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling ATG7 with purified ab133528 at 1/150 dilution (8.5μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1/1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• WB

Lab

Western blot - Anti-ATG7 antibody [EPR6251] (AB133528)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ATG7 antibody [EPR6251] (ab133528) at 1/50000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 2:

Mouse spleen lysate at 20 µg

Lane 3:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 4:

Mouse kidney lysate at 20 µg

Lane 5:

Rat kidney 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: 47 kDa,77 kDa

Observed band size: 47 kDa,77 kDa

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• WB

Lab

Western blot - Anti-ATG7 antibody [EPR6251] (AB133528)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : ATG7 knockout HAP1 cell lysate (20 μg)
Lane 3 : Jurkat cell lysate (20 μg)
Lane 4 : HepG2 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab133528 observed at 77 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab133528 was shown to specifically react with ATG7 when ATG7 knockout samples were used. Wild-type and Apg7 knockout samples were subjected to SDS-PAGE. ab133528 and ab8245 (loading control to GAPDH) were diluted 1/10,000 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ATG7 antibody [EPR6251] (ab133528)

Predicted band size: 77 kDa

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• WB

Lab

Western blot - Anti-ATG7 antibody [EPR6251] (AB133528)

False colour image of Western blot : Anti-ATG7 antibody [EPR6251] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab133528 was shown to bind specifically to ATG7. A band was observed at 75 kDa in wild-type HeLa cell lysates with no signal observed at this size in ATG7 knockout cell line ab283307 (knockout cell lysate ab287353). To generate this image, wild-type and ATG7 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-ATG7 antibody [EPR6251] (ab133528) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ATG7 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ATG7 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-atg7-knockout-hela-cell-line-ab283307'>ab283307</a>)

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Predicted band size: 77 kDa

Observed band size: 75 kDa

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• WB

Lab

Western blot - Anti-ATG7 antibody [EPR6251] (AB133528)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-ATG7 antibody [EPR6251] (ab133528) at 1/10000 dilution

Lane 1:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Rat spleen 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: 77 kDa

Observed band size: 77 kDa

false