All lanes : Anti-PARK7/DJ1 antibody [EP2815Y] (ab76008) 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?

This data was developed using ab76008, the same antibody clone in a different buffer formulation.

Lanes 1-4: Merged signal (red and green). Green - ab76008 observed at 24 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab76008 Anti-PARK7/DJ1 antibody [EP2815Y] 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. ab76008 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.

This IHC data was generated using the same anti-PARK7 antibody clone, EP2815Y, in a different buffer formulation (cat# ab76008).

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling PARK7/DJ1 with Purified ab76008 at 1:500 dilution (0.2 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This IHC data was generated using the same anti-PARK7 antibody clone, EP2815Y, in a different buffer formulation (cat# ab76008).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung cancer tissue sections labeling PARK7/DJ1 with Purified ab76008 at 1:1000 dilution (0.11 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

This IHC data was generated using the same anti-PARK7 antibody clone, EP2815Y, in a different buffer formulation (cat# ab76008).

 

Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling PARK7/DJ1 with Purified ab76008 at 1:20 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

This WB data was generated using the same anti-PARK7 antibody clone, EP2815Y, in a different buffer formulation (cat# ab76008).

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 - ab76008 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab76008 was shown to specifically react with PARK/DJ1 when PARK/DJ1 knockout samples were used. Wild-type and PARK/DJ1 knockout samples were subjected to SDS-PAGE. ab76008 and ab8245 (loading control to GAPDH) 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 1 h at room temperature before imaging.

This IHC data was generated using the same anti-PARK7 antibody clone, EP2815Y, in a different buffer formulation (cat# ab76008).

ab76008 (purified) at 1:20 dilution (0.5µg) immunoprecipitating PARK7/DJ1 in Mouse brain lysate.
Lane 1 (input): Mouse brain lysate 10µg
Lane 2 (+): ab76008 & Mouse brain lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76008 in Mouse brain lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunocytochemistry/Immunofluorescence analysis of MCF-7 (human breast carcinoma) labelling PARK7/DJ1 with purified ab76008 at 1/500. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Control: PBS only

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76008).

This image was generated using the unpurified version of the product. 

Overlay histogram showing HepG2 cells stained with ab76008 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab76008, 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 HepG2 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76008).

This image was generated using the unpurified version of the product. 

This IHC data was generated using the same anti-PARK7 antibody clone, EP2815Y, in a different buffer formulation (cat# ab76008).

ab76008, at 1/250 dilution, staining PARK7/DJ1 in human brain by immunohistochemistry using paraffin-embedded tissue.

This image was generated using the unpurified version of the product. 

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.