Anti-CEBP Alpha/CEBPA antibody [EP708Y] (ab40761) at 1/1000 dilution + U937 + LPS cell lysate at 10 µg

Predicted band size: 43 kDa
Observed band size: 30,43 kDa why is the actual band size different from the predicted?

We are unsure as to the identity of the 30kda band.

ab40761 (1/250) staining HeLa cells using immunofluorescence.

Overlay histogram showing HeLa cells stained with ab40761 (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 (ab40761, 1/100 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 IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Anti-CEBP Alpha/CEBPA antibody [EP708Y] (ab40761) at 1/5000 dilution + Human MCF-7 whole cell lysate at 100000 cells

Secondary
HRP-conjugated Donkey anti-rabbit IgG polyclonal at 1/2000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 43 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

Blocked with 5% Milk for 1 hour at 24°C.

We are unsure as to the identity of the 30kda band.

See Abreview