Anti-ATG7 antibody [EP1759Y] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ATG7 antibody [EP1759Y] - BSA and Azide free (AB227564)

Overlay histogram showing HEK293 cells stained with unpurified ab52472 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52472, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 100% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52472).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ATG7 antibody [EP1759Y] - BSA and Azide free (AB227564)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling ATG7 with purified ab52472 at 1/100. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab7291 anti-Tubulin (mouse mAb) followed by ab150120, AlexaFluor^®594 goat anti-mouse secondary both at 1/1000. Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody was used followed by anti-mouse secondary antibody (ab150120). For negative control 2, ab7291 (mouse primary antibody) was used followed by anti-rabbit secondary antibody (ab150077).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52472).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ATG7 antibody [EP1759Y] - BSA and Azide free (AB227564)

Intracellular Flow Cytometry analysis of HeLa cells labelling ATG7 (red) with purified ab52472 at dilution of 1/100. The secondary antibody used was goat anti rabbit IgG (FITC) at 1/500. Cells were fixed with 4% paraformaldehyde. Isotype control antibody was Rabbit monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52472).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG7 antibody [EP1759Y] - BSA and Azide free (AB227564)

This IHC data was generated using the same anti-ATG7 antibody clone, EP1759Y, in a different buffer formulation (cat# ab52472.

Immunohistochemical analysis of paraffin-embedded human cervical carcinoma sections labelling Apg7 with purified ab52472 at a dilution of 1/500. The secondary antibody used was ab97051, Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 1/500. The sample was counterstained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ATG7 antibody [EP1759Y] - BSA and Azide free (AB227564)

Immunocytochemistry/Immunofluorescence analysis of HT-29 (human colorectal adenocarcinoma) cells labelling ATG7 with purified ab52472 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei couterstained with DAPI (blue).

Secondary Only Control : 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 (ab52472).

• IP

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Immunoprecipitation - Anti-ATG7 antibody [EP1759Y] - BSA and Azide free (AB227564)

ab52472 at 1/30 dilution immunoprecipitating ATG7 in HEK293 whole cell lysate observed at 70 KDa (lanes 1 and 2).

Lane 1 (input) : HEK293 whole cell lysate 10ug

Lane 2 (+) : ab52472 + HEK293 whole cell lysate

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab52472 in HEK293 whole cell lysate

For western blotting, ab52472 was used followed by VeriBlot for IP Detection Reagent (HRP) (ab131366) for detection at 1/10,000 dilution .

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52472).

All lanes:

Immunoprecipitation - Anti-ATG7 antibody [EP1759Y] (<a href='/en-us/products/primary-antibodies/atg7-antibody-ep1759y-ab52472'>ab52472</a>)

Predicted band size: 49 kDa,77 kDa

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• WB

Lab

Western blot - Anti-ATG7 antibody [EP1759Y] - BSA and Azide free (AB227564)

False colour image of Western blot : Anti-ATG7 antibody [EP1759Y] staining at 1/100000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab52472 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 [EP1759Y] (<a href='/en-us/products/primary-antibodies/atg7-antibody-ep1759y-ab52472'>ab52472</a>) at 1/100000 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

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Western blot - Anti-ATG7 antibody [EP1759Y] - BSA and Azide free (AB227564)

This WB data was generated using the same anti-ATG7 antibody clone, EP1759Y, in a different buffer formulation (cat# ab52472).

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : Apg7 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 - ab52472 observed at 77 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab52472 was shown to specifically react with Apg7 when Apg7 knockout samples were used. Wild-type and ProteinX knockout samples were subjected to SDS-PAGE. ab52472 and ab8245 (loading control to Apg7) were both diluted 1/2000 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ATG7 antibody [EP1759Y] (<a href='/en-us/products/primary-antibodies/atg7-antibody-ep1759y-ab52472'>ab52472</a>)

Predicted band size: 77 kDa

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