Anti-Ndufs4 antibody [EP7832] (ab137064) at 1/50000 dilution (purified) + Rat heart lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 20 kDa

Blocking and diluting buffer: 5% NFDM/TBST

ab137064 (purified) at 1:30 dilution (2μg) immunoprecipitating Ndufs4 in Rat heart lysate.

Lane 1 (input): Rat heart lysate, 10μg
Lane 2 (+): ab137064 & Rat heart lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab137064 in Rat heart 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.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Ndufs4 with purified ab137064 at 1:60 dilution (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor ® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Ndusf5 with Purified ab137064 at 1:500 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab7291 anti-Tubulin (mouse mAb) ab150120 AlexaFluor ® 594 Goat anti-Mouse secondary (1:1000,2 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor ® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebrum tissue sections labeling Ndufs4 with Purified ab137064 at 1:50 dilution. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling Ndufs4 with Purified ab137064 at 1:50 dilution. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human stomach tissue sections labeling Ndufs4 with Purified ab137064 at 1:50 dilution. Heat mediated antigen retrieval was performed using Tris/EDTA buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-Ndufs4 antibody [EP7832] (ab137064) at 1/2000 dilution (purified)

Lane 1 : Human fetal brain lysates
Lane 2 : Mouse heart lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 20 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Ndufs4 knockout HAP1 cell lysate (20 µg)
Lane 3: HEK293 cell lysate (20 µg)
Lane 4: Human fetal heart tissue lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab137064 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab137064 was shown to recognize Ndufs4 when Ndufs4 knockout samples were used, along with additional cross-reactive bands. Wild-type and Ndufs4 knockout samples were subjected to SDS-PAGE. ab137064 and ab8245 (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-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.

Immunohistochemical analysis of paraffin-embedded Human brain tissue labelling Ndufs4 with unpurified ab137064 at 1/50 dilution.

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

Overlay histogram showing HepG2 cells stained with unpurified ab137064 (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 (ab137064, 1/1000 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) (0.1μg/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 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-Ndufs4 antibody [EP7832] (ab137064) at 1/1000 dilution (unpurified)

Lane 1 : 293T cell lysate
Lane 2 : Fetal brain tissue lysate
Lane 3 : Fetal kidney tissue lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 20 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labelling Ndufs4 with unpurified ab137064 at 1/50 dilution.

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