The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 - 5 µg/ml. Predicted molecular weight: 84 kDa.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 5 µg/ml.
Use 1µg for 106 cells. ab170191-Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
Use at an assay dependent concentration.
FunctionEssential transmembrane GTPase, which mediates mitochondrial fusion. Fusion of mitochondria occurs in many cell types and constitutes an important step in mitochondria morphology, which is balanced between fusion and fission. MFN1 acts independently of the cytoskeleton. Overexpression induces the formation of mitochondrial networks.
Tissue specificityUbiquitous. Expressed at slightly higher level in kidney and heart. Isoform 2 may be overexpressed in some tumors, such as lung cancers.
Sequence similaritiesBelongs to the mitofusin family.
Post-translational modificationsUbiquitinated by MARCH5.
Cellular localizationCytoplasm and Mitochondrion outer membrane.
Immunocytochemistry/ Immunofluorescence - Anti-Mitofusin 1 antibody (ab57602)This image is courtesy of an anonymous Abreview
ab57602 staining Mitofusin 1 in the H9C2 cell line from Rat cardiomyocytes by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 1% and blocked with 5% serum for 60 minutes at 21°C. Samples were incubated with primary antibody (1/200 in PBS + 5% Goat serum) for 2 hours at 21°C. An Alexa Fluor®488-conjugated Goat anti-mouse IgG polyclonal(1/400) was used as the secondary antibody.
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.
IHC image of ab57602 staining in human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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.
Flow Cytometry-Mitofusin 1 antibody(ab57602)
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.
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 70oC; 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.
References for Anti-Mitofusin 1 antibody (ab57602)
This product has been referenced in:
MacVicar TD & Lane JD Impaired OMA1-dependent cleavage of OPA1 and reduced DRP1 fission activity combine to prevent mitophagy in cells that are dependent on oxidative phosphorylation. J Cell Sci127:2313-25 (2014).
Read more (PubMed: 24634514) »
Yun J et al. MUL1 acts in parallel to the PINK1/parkin pathway in regulating mitofusin and compensates for loss of PINK1/parkin. Elife (Cambridge)3:e01958 (2014).
Read more (PubMed: 24898855) »