Anti-HPRT antibody (ab10479)

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: HPRT1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: A431 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab10479 observed at 25 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab10479 was shown to specifically react with HPRT1 in wild-type HAP1 cells. No band was observed when HPRT1 knockout samples were examined. Wild-type and HPRT1 knockout samples were subjected to SDS-PAGE.  Ab10479 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/500 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.

ICC/IF image of ab10479 stained human HeLa cells. The cells were methanol fixed ( 5 min) and incubated with the antibody (ab10479, 5µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 phalloidin was used to label F-actin (red).