Recombinant Anti-Src antibody [EGTR103] (ab133283)

False colour image of Western blot: Anti-Src antibody [EGTR103] 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, ab133283 was shown to bind specifically to Src. A band was observed at 65 kDa in wild-type HAP1 cell lysates with no signal observed at this size in SRC knockout cell line HAP1. To generate this image, wild-type and SRC knockout HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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: Src knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: A431 knockout HAP1 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab133283 observed at 60 kDa. Red - loading control, ab8226, observed at 42 kDa.
Unpurified ab133283 was shown to specifically react with Src when Src knockout samples were used. Wild-type and Src knockout samples were subjected to SDS-PAGE. ab133283 and ab8226 (loading control to beta actin) were both diluted 1/1000 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 1/10000 dilution for 1 h at room temperature before imaging.