Recombinant Anti-Ndufs4 antibody [EP7832] (ab137064)

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.

Intracellular 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).

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 withunpurified 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. 

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.