Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Drebrin knockout HAP1 whole cell lysate (20 µg)
Lane 3: SH-SY5Y whole cell lysate (20 µg)

Lanes 1 - 3: Merged signal (red and green). Green - ab60932 observed at 120 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab60932 was shown to specifically react with Drebrin in wild-type cells as signal was lost in Drebrin knockout cells. Wild-type and Drebrin knockout samples were subjected to SDS-PAGE. Ab60932 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 5 µg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Drebrin antibody [mAbcam60932] (ab60932) at 10 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 120 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?
Additional bands at: 10 kDa, 65 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 8 minutes


Ab60932 detects a band around 110-kDa. While this differs to its predicted molecular weight of 71-kDa, it is consistent with what has been described in the literature(PMID:10604653).

All lanes : Anti-Drebrin antibody [mAbcam60932] (ab60932) at 10 µg/ml

Lane 1 : Human Cortex Neuronal cell lysate

Lane 2 : Rat Cortex Neuronal cell lysate
Lane 3 : Rat Hippocampus Tissue Lysate
Lane 4 : Human skin tumor tissue lysate (melanoma) - total protein (ab29339)
Lane 5 : Malme 3M (Human melanoma cells) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Predicted band size: 120 kDa
Observed band size: 100,120 kDa why is the actual band size different from the predicted?
Additional bands at: 200 kDa, 45 kDa, 50 kDa, 60 kDa. We are unsure as to the identity of these extra bands.

Overlay histogram showing SHSY-5Y cells stained with ab60932 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab60932, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SHSY-5Y cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.