Recombinant Anti-RIP antibody [EPR4689] (ab125072)

False colour image of Western blot: Anti-RIP antibody [EPR4689] 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, ab125072 was shown to bind specifically to RIP. A band was observed at 76 kDa in wild-type THP-1 cell lysates with no signal observed at this size in RIPK1 knockout cell line ab276121 (knockout cell lysate ab284210). To generate this image, wild-type and RIPK1 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 cell lysate (20 µg)
Lane 2: RIP knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Raji cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab125072 (unpurified) observed at 78 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab125072 was shown to specifically react with RIP in wild-type HAP1 cells. No band was observed when RIP knockout samples were examined. Wild-type and RIP knockout samples were subjected to SDS-PAGE. ab125072 at a dilution of 1/1000 and ab8245 (loading control to GAPDH)  at a dilution of 1/10,000 were 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 hour at room temperature before imaging.