Anti-APG5L/ATG5 antibody [EPR4797]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-APG5L/ATG5 antibody [EPR4797] (AB109490)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling APG5L/ATG5 with purified ab109490 at 1/250 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^®488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-APG5L/ATG5 antibody [EPR4797] (AB109490)

ab109490, at 1/100 dilution, staining APG5L/ATG5 in paraffin-embedded Human breast carcinoma by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-APG5L/ATG5 antibody [EPR4797] (AB109490)

ab109490, at 1/100 dilution, staining APG5L/ATG5 in paraffin-embedded Human kidney by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-APG5L/ATG5 antibody [EPR4797] (AB109490)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : APG5L/ATG5 knockout HAP1 cell lysate (20 μg)
Lane 3 : Raji cell lysate (20 μg)
Lane 4 : Jeg-3 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab109490 observed at 52 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109490 was shown to specifically react with APG5L/ATG5 when APG5L/ATG5 knockout samples were used. Wild-type and APG5L/ATG5 knockout samples were subjected to SDS-PAGE. ab109490 and ab8245 (loading control to GAPDH) were diluted 1/1000 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-APG5L/ATG5 antibody [EPR4797] (ab109490)

Predicted band size: 32 kDa

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• WB

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Western blot - Anti-APG5L/ATG5 antibody [EPR4797] (AB109490)

Upper band shows APG5L/ATG5 conjugated to ATG12.

All lanes:

Western blot - Anti-APG5L/ATG5 antibody [EPR4797] (ab109490) at 1/1000 dilution

All lanes:

Human fetal kidney lysate at 10 µg

Predicted band size: 32 kDa

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• WB

Lab

Western blot - Anti-APG5L/ATG5 antibody [EPR4797] (AB109490)

False colour image of Western blot : Anti-APG5L/ATG5 antibody [EPR4797] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109490 was shown to bind specifically to APG5L/ATG5. A band was observed at 50 kDa in wild-type THP-1 cell lysates with no signal observed at this size in ATG5 knockout cell line ab277835 (knockout cell lysate ab290722). To generate this image, wild-type and ATG5 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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.

All lanes:

Western blot - Anti-APG5L/ATG5 antibody [EPR4797] (ab109490) at 1/1000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

ATG5 knockout THP-1 cell lysate at 20 µg

Lane 3:

U-87 MG cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 50 kDa

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• WB

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Western blot - Anti-APG5L/ATG5 antibody [EPR4797] (AB109490)

All lanes:

Western blot - Anti-APG5L/ATG5 antibody [EPR4797] (ab109490) at 1/1000 dilution

Lane 1:

THP1 cell lysate at 10 µg

Lane 2:

Raji cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

HT1080 cell lysate at 10 µg

Predicted band size: 32 kDa

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