Anti-Mitofusin 2 antibody [EPR19796]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

Flow cytometry overlay histogram showing left wild-type HEK293 positive cells and right negative MFN2 knockout HEK293 stained with ab205236 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab205236) (1x 10⁶ in 100μl at 0.04 μg/ml (1/18525)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Isotype control antibody was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (black line) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

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 Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

Intracellular 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 Fluorr^® 488) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

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.

All lanes:

Immunoprecipitation - Anti-Mitofusin 2 antibody [EPR19796] (ab205236)

Predicted band size: 86 kDa

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• WB

Lab

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

Western blot : Anti-MFN2 antibody [EPR19796] (ab205236) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab205236 was shown to bind specifically to MFN2. A band was observed at 80 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in MFN2 knockout cell line. To generate this image, wild-type and MFN2 knockout HEK-293 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (ab205236) at 1/2000 dilution

Lane 1:

Wild-type HEK-293 cell lysate at 20 µg

Lane 2:

MFN2 knockout HEK-293 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

PC-3 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 80 kDa

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• WB

Supplier Data

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (ab205236) at 1/2000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 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 1/100000 dilution

Predicted band size: 86 kDa

Observed band size: 86 kDa

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Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

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:

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (ab205236) at 1/5000 dilution

Lane 1:

Human Mitofusin 1 recombinant protein fragment at 0.01 µg

Lane 2:

Human Mitofusin 2 recombinant protein fragment at 0.01 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 1/100000 dilution

Predicted band size: 86 kDa

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Exposure time: 1s

• WB

Supplier Data

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

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:

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (ab205236) at 1/5000 dilution

Lane 1:

Human fetal heart lysate at 10 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Lane 3:

Human fetal liver lysate at 10 µg

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

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• WB

Lab

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (AB205236)

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:

Western blot - Anti-Mitofusin 2 antibody [EPR19796] (ab205236) at 1/2000 dilution

Lane 1:

Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

MFN2 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human MFN2 (Mitofusin 2) knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-mfn2-mitofusin-2-knockout-hek-293-cell-line-ab260861'>ab260861</a>)

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Predicted band size: 86 kDa

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