All lanes : Anti-NDUFB9 antibody [EPR15955-78] (ab200198) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : NDUFB9 knockout HeLa cell lysate
Lane 3 : Jurkat cell 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: 22 kDa
Observed band size: 22 kDa

Lanes 1-3: Merged signal (red and green). Green - ab200198 observed at 22 kDa. Red - loading control ab8245 observed at 36 kDa.

ab200198 Anti-NDUFB9 antibody [EPR15955-78] was shown to specifically react with NDUFB9 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265946 (knockout cell lysate ab258065) was used. Wild-type and NDUFB9 knockout samples were subjected to SDS-PAGE. ab200198 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.

 

All lanes : Anti-NDUFB9 antibody [EPR15955-78] (ab200198) at 1/5000 dilution

Lane 1 : Jurkat (Human T cell leukemia cells from peripheral blood) cell lysate
Lane 2 : HEK293 (Human embryonic kidney) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-NDUFB9 antibody [EPR15955-78] (ab200198) at 1/5000 dilution + Human fetal liver lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-NDUFB9 antibody [EPR15955-78] (ab200198) at 1/1000 dilution

Lane 1 : Mouse kidney lysate
Lane 2 : Mouse spleen lysate
Lane 3 : Rat heart lysate
Lane 4 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 22 kDa
Observed band size: 22 kDa


Exposure time: 1 minute

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling NDUFB9 with ab200198 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Cytoplasm staining on Human kidney tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse cardiac muscle tissue labeling NDUFB9 with ab200198 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution.

Cytoplasm staining on mouse cardiac muscle tissue is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling NDUFB9 with ab200198 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green).

Cytoplasm staining on Jurkat cell line is observed.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab200198 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling NDUFB9 with ab200198 at 1/400 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

NDUFB9 was immunoprecipitated from 1mg of Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate with ab200198 at 1/150 dilution.

Western blot was performed from the immunoprecipitate using ab200198 at 1/1000 dilution.

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: Jurkat whole cell lysate 10 µg (Input).

Lane 2: ab200198 IP in Jurkat whole cell elysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab200198 in Jurkat whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.