重组Anti-ATG12抗体[EPR4800] (ab109491)

All lanes : Anti-ATG12 antibody [EPR4800] (ab109491) at 1/1000 dilution

Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : ATG12 knockout THP-1 cell lysate
Lane 3 : HCT 116 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 15 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?

False colour image of Western blot: Anti-ATG12 antibody [EPR4800] 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, ab109491 was shown to bind specifically to ATG12. A band likely to be the unfunctional complex with ATG5 was observed at 52 kDa in wild-type THP-1 cell lysates with no signal observed at this size in Atg12 knockout cell line ab277831 (knockout cell lysate ab278183) - unconjugated functional form not observed at 15 kDa. To generate this image, wild-type and Atg12 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 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.

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: ATG12 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HCT116 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab109491 observed at 55 kDa. Observed band shows ATG12-ATG5 conjugation. Red - loading control, ab8245, observed at 37 kDa.

Ab109491 was shown to specifically react with

ATG12 in wild-type cells along with additional cross-reactive bands. The band was not seen in ATG12 knockout HAP1 cells. Wild-type and ATG12 knockout samples were subjected to SDS-PAGE.  Ab109491 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-ATG12 antibody [EPR4800] (ab109491) at 1/1000 dilution

Lane 1 : Human fetal kidney tissue lysate
Lane 2 : HepG2 cell lysate
Lane 3 : Raji lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 15 kDa

Observed band shows ATG12-ATG5 conjugation

All lanes : Anti-ATG12 antibody [EPR4800] (ab109491) at 1/1000 dilution

Lane 1 : ATG12 transfected 293T lysate
Lane 2 : Non-transfected 293T lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 15 kDa

ab109491, at 1/250 dilution, staining ATG12 in paraffin-embedded Human breast carcinoma by Immunohistochemistry.

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