All lanes : Anti-PARK7/DJ1 antibody [EP2816Y] (ab76241) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : PARK7 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : Human brain nuclear fraction tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 20 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab76241 observed at 24 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab76241 Anti-PARK7/DJ1 antibody [EP2816Y] was shown to specifically react with PARK7/DJ1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266338 (knockout cell lysate ab257016) was used. Wild-type and PARK7/DJ1 knockout samples were subjected to SDS-PAGE. ab76241 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing Jurkat cells stained with ab76241 (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 (ab76241, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PARK/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 - ab76241 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab76241 was shown to specifically react with PARK7/DJ1 in wild-type HAP1 cells. No band was observed when PARK7/DJ1 knockout samples were used. Wild-type and PARK7/DJ1 knockout samples were subjected to SDS-PAGE. ab76241 and ab8245 (loading control to PARK7/DJ1) were both diluted 1/10,000 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 1/10,000 dilution for 1hr at room temperature before imaging.

All lanes : Anti-PARK7/DJ1 antibody [EP2816Y] (ab76241) at 1/20000 dilution

Lane 1 : TF-1 cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/1000 dilution

Predicted band size: 20 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded human brain tissue using ab76241 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.