重组Anti-FTO抗体[EPR6895] (ab124892)

False colour image of Western blot: Anti-FTO antibody [EPR6895] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab124892 was shown to bind specifically to FTO. A band was observed at 58 kDa in wild-type MCF7 cell lysates with no signal observed at this size in FTO knockout cell line. To generate this image, wild-type and FTO knockout MCF7 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: FTO knockout HAP1 whole cell lysate (20 µg)
Lane 3: HEK293 whole cell lysate (20 µg)
Lane 4: MOLT4 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab124892 observed at 58 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab124892 was shown to specifically react with FTO in wild-type HAP1 cells. No band was observed when FTO knockout samples were examined. Wild-type and FTO knockout samples were subjected to SDS-PAGE. ab124892 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/1000 dilution and 1/10,000 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung carcinoma tissue sections labeling FTO with purified ab124892 at 1:100 dilution (1.66 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast tissue sections labeling FTO with purified ab124892 at 1:100 dilution (1.66 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

ab124892, at 1/100 dilution, staining FTO in Paraffin-embedded Human Breast tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

ab124892, at 1/100 dilution, staining FTO in Paraffin-embedded Human kidney tissue by Immunohistochemistry. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.