All lanes : Anti-Mitofusin 2 antibody [EPR19796] (ab205236) at 1/2000 dilution

Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : MFN2 knockout HEK-293 whole cell lysate
Lane 3 : Jurkat whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 86 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab205236 observed at 86 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab205236 was shown to recognize MFN2 (Mitofusin 2) in wild-type HEK-293 cells as signal was lost at the expected MW in MFN2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and MFN2 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab205236 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Mitofusin 2 antibody [EPR19796] (ab205236) at 1/5000 dilution

Lane 1 : Human Mitofusin 1 recombinant protein fragment
Lane 2 : Human Mitofusin 2 recombinant protein fragment

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 86 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

Human Mitofusin 1 recombinant protein fragment contains aa130-485 with a His-Tag®. Human Mitofusin 2 recombinant protein fragment contains aa151-506 with a His-Tag®.

All lanes : Anti-Mitofusin 2 antibody [EPR19796] (ab205236) at 1/5000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate
Lane 3 : Human fetal liver lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 86 kDa
Observed band size: 86 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1: 15 seconds; Lane 2 and 3: 30 seconds.

The expression profile is consistent with the literature (PMID 14561718; PMID 25574749).

All lanes : Anti-Mitofusin 2 antibody [EPR19796] (ab205236) at 1/2000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 86 kDa
Observed band size: 86 kDa


Exposure time: 10 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Mitofusin 2 with ab205236 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing mitochondrial staining on HeLa cell line.

The nuclear counter stain is DAPI (blue). COX IV is detected with ab33985 (Anti-COX IV antibody [mAbcam33985] - Mitochondrial Marker), at 1/200 dilution, followed by secondary detection using ab150120 Alexa Fluor^® 594 Goat anti-Mouse (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Mitofusin 2 with ab205236 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/200 dilution, followed by secondary detection using ab150120 Alexa Fluor^® 594 Goat anti-Mouse (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Mitofusin 2 with ab205236 at 1/700 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 (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Mitofusin 2 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab205236 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab205236 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

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

Lane 2: ab205236 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab205236 in HeLa whole cell lysate.

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

Exposure time: 3 seconds.