All lanes : Anti-MEF2A antibody [EP1706Y] (ab76063) at 0.2 µg/ml (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : 3T3-L1 (Mouse embryonic fibroblast) whole cell lysates
Lane 3 : Mouse brain lysates
Lane 4 : Rat brain lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 55 kDa
Observed band size: 66 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST.

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling MEF2A with Purified ab76063 at 1:100 dilution (4.3 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor®594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor®488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Overlay histogram showing MCF-7 cells stained with unpurified ab76063 (red line). The cells were fixed with 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 (ab76063, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF-7 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

All lanes : Anti-MEF2A antibody [EP1706Y] (ab76063) at 1/10000 dilution (Unpurified)

Lane 1 : Fetal brain lysate
Lane 2 : Human heart lysate
Lane 3 : HeLa lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 55 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?



The observed band size may be different from the predicted molecular weight as the protein is sumoylated.

All lanes : Anti-MEF2A antibody [EP1706Y] (ab76063) at 1/1000 dilution (Unpurified)

All lanes : Murine cardiomyocyte whole cell lysate

Lysates/proteins at 25 µg per lane.

Secondary
All lanes : Goat anti-rabbit polyclonal HRP conjugate.

Performed under reducing conditions.

Predicted band size: 55 kDa


Exposure time: 30 seconds

Lanes from left to right: cropped MW marker ladder, 1, 2, 3, 4.

Blocking was performed with 5% milk for 1 hour at room temperature.

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Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling MEF2A with purified ab76063 at 1:40 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).