All lanes : Anti-Mitofusin 2 + Mitofusin 1 antibody [3C9] (ab57602) at 1/1000 dilution

Lane 1 : Recombinant Human Mitofusin 1 protein (ab132635)
Lane 2 : MFN2 OE lysate (DDK tag) 10ng

Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/10000 dilution

Predicted band size: 84 kDa

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab57602 overnight at 4°C. Antibody binding was detected using the Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 at a 1/10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

The difference in MW is due to lane one having a GST tag and lane 2 a DDK tag.

ICC/IF image of ab57602 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab57602, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

This image was generated using a version of the antibody produced in ascites. 

All lanes : Anti-Mitofusin 2 + Mitofusin 1 antibody [3C9] (ab57602) at 1/1000 dilution

All lanes : Mouse cardiomyocytes whole cell lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-mouse IgG at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 84 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocked with 5% milk for 1 hour at 25°C.

Incubated with the primary antibody at 4°C for 13 hours in 1X TBS.

This image was generated using a version of the antibody produced in ascites. 

See Abreview

Mitofusin 2 + Mitofusin 1 antibody (ab57602) at 1ug/lane + HeLa cell lysate at 25 μg/lane.

This image was generated using a version of the antibody produced in ascites. 

IHC image of ab57602 staining in human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab57602, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

This image was generated using a version of the antibody produced in ascites. 

Overlay histogram showing HEK293 cells stained with ab57602 (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 (ab57602, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This image was generated using a version of the antibody produced in ascites. 

Mitofusin 2 + Mitofusin 1 was immunoprecipitated using 0.5mg Hela whole cell extract, 10ug of Mouse monoclonal to Mitofusin 1 and 50µl of protein G magnetic beads (lane 1). The antibody was incubated with the Protein G beads for 10 min under agitation. No antibody was added to the control lane 2 and no extract or antibody was added to contol lane 3. Hela whole cell extract diluted in RIPA buffer was added to each sample and incubated for 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab57602.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Bands: 84kDa:Mitofusin 1; 60kDa bead background: non specific - 48kDa: We are unsure as to the identity of this extra band.

This image was generated using a version of the antibody produced in ascites.