All lanes : Anti-Mitofusin 2 + Mitofusin 1 antibody [EPR19792] (ab205235) at 1/2000 dilution

Lane 1 : Human Mitofusin 2 recombinant protein fragment
Lane 2 : Human Mitofusin 1 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: 84, 86 kDa


Exposure time: 1 second

Blocking/Dilution buffer: 5% NFDM/TBST.

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

All lanes : Anti-Mitofusin 2 + Mitofusin 1 antibody [EPR19792] (ab205235) at 1/1000 dilution

Lane 1 : Human fetal kidney lysate
Lane 2 : Human fetal liver lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 84, 86 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1 and 2: 15 seconds; Lane 3: 3 seconds.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Mitofusin 2 + Mitofusin 1 with ab205235 at 1/500 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 cells.

The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (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 + Mitofusin 1 with ab205235 at 1/500 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 cells.

The nuclear counter stain is DAPI (blue). Cox IV is detected with ab33985 (anti-Cox IV mouse mAb) at 1/1000 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab205235 at 1/500 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab33985 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Mitofusin 2 + Mitofusin 1 with ab205235 at 1/50 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A]-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 + Mitofusin 1 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab205235 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab205235 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: ab205235 IP in HeLa whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab205235 in HeLa whole cell lysate.

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

Exposure time: 5 seconds.