Anti-HIF-2-alpha antibody [OTI2G5] (ab157249)

False colour image of Western blot: Anti-HIF-2-alpha antibody [OTI2G5] staining at 1/500 dilution, shown in black; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab157249 was shown to bind specifically to HIF-2-alpha. A band was observed at 100 kDa in treated wild-type A549 cell lysates with no signal observed at this size in EPAS1 knockout cell line ab259774 (knockout cell lysate ab259779). To generate this image, wild-type and EPAS1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % BSA 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 before development with Optiblot (ECL reagent ab133456) and imaged with 20 seconds exposure time. Secondary antibodies used were HRP conjugated Goat anti-Mouse (H+L) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.