Anti-Ndufs4 antibody [EP7832]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ndufs4 antibody [EP7832] (AB137064)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Ndufs4 antibody [EP7832] (AB137064)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells (human cervix adenocarcinoma epithelial cells) labeling Ndusf4 with purified ab137064 at a 1/500 dilution. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% tritonX-100.

Cells were counterstained with ab7291 Anti-alpha Tubulin antibody [DM1A] - Loading Control followed by ab150120 Alexa Fluor^® 594 Goat anti-Mouse secondary at a 1/1000 dilution (2 µg/ml). ab150077 Goat anti-rabbit IgG (Alexa Fluor^® 488) was used as the secondary antibody at 1/1000 dilution. DAPI nuclear counterstain.

A secondary antibody-only control was performed by omitting the primary antibody and using PBS instead.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab137064).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ndufs4 antibody [EP7832] (AB137064)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Ndufs4 antibody [EP7832] (AB137064)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ndufs4 antibody [EP7832] (AB137064)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ndufs4 antibody [EP7832] (AB137064)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ndufs4 antibody [EP7832] (AB137064)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ndufs4 antibody [EP7832] (AB137064)

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.

• IP

Lab

Immunoprecipitation - Anti-Ndufs4 antibody [EP7832] (AB137064)

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.

All lanes:

Immunoprecipitation - Anti-Ndufs4 antibody [EP7832] (ab137064)

Predicted band size: 20 kDa

false

• WB

Lab

Western blot - Anti-Ndufs4 antibody [EP7832] (AB137064)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Ndufs4 antibody [EP7832] (ab137064) at 1/2000 dilution

Lane 1:

Human fetal brain lysates at 15 µg

Lane 2:

Mouse heart lysates at 15 µg

Secondary

All lanes:

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

Predicted band size: 15 kDa,20 kDa,87 kDa,97 kDa

Observed band size: 18 kDa

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• WB

Lab

Western blot - Anti-Ndufs4 antibody [EP7832] (AB137064)

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.

All lanes:

Western blot - Anti-Ndufs4 antibody [EP7832] (ab137064)

Predicted band size: 20 kDa

false

• WB

Unknown

Western blot - Anti-Ndufs4 antibody [EP7832] (AB137064)

All lanes:

Western blot - Anti-Ndufs4 antibody [EP7832] (ab137064) at 1/1000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

Fetal brain tissue lysate at 10 µg

Lane 3:

Fetal kidney tissue lysate at 10 µg

Predicted band size: 20 kDa

Observed band size: 18 kDa

false

• WB

Lab

Western blot - Anti-Ndufs4 antibody [EP7832] (AB137064)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Ndufs4 antibody [EP7832] (ab137064) at 1/50000 dilution

All lanes:

Rat heart lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 20 kDa

false