Western blot using ab11251 at 1/500 on HeLa whole cell lysate (20µg/lane).

Western blot using ab11251 at 1/500 on HeLa whole cell lysate (20µg/lane).

Western blot using clone malphaDJ-1/E2.19 and a beta actin antibody as a loading control.

The bottom band is PARK7/DJ1, the top band is beta actin.

Lane 1: 293 cell lysate
Lane 2: MCF-7 cell lysate
Lanes 3-7: various different prostate cell lines
Lane 8: recombinant PARK7/DJ1 (that was used as immunogen for this antibody)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PARK7/DJ1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Human brain tissue lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab11251  observed at 24 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab11251 was shown to specifically react with PARK7/DJ1 in wild-type HAP1 cells. No band was observed when knockout samples were used. Wild-type and PARK7/DJ1 knockout samples were subjected to SDS-PAGE. ab11251 and ab181602 (loading control to GAPDH) were diluted at 1/500 and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging.

ICC/IF image of ab11251 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab11251, 1:500 dilution) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

Overlay histogram showing HepG2 cells stained with ab11251 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab11251, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgM [ICIGM] (ab91545, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.