Anti-ATG7 antibody [EPR6251] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ATG7 antibody [EPR6251] - BSA and Azide free (AB232348)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133528).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ATG7 antibody [EPR6251] - BSA and Azide free (AB232348)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133528).

• WB

Lab

Western blot - Anti-ATG7 antibody [EPR6251] - BSA and Azide free (AB232348)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab133528).

All lanes:

Western blot - Anti-ATG7 antibody [EPR6251] (<a href='/en-us/products/primary-antibodies/atg7-antibody-epr6251-ab133528'>ab133528</a>)

Predicted band size: 77 kDa

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• WB

Lab

Western blot - Anti-ATG7 antibody [EPR6251] - BSA and Azide free (AB232348)

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] (<a href='/en-us/products/primary-antibodies/atg7-antibody-epr6251-ab133528'>ab133528</a>) 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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