Anti-CAB39/MO25 antibody [EP1680Y] (ab51132) at 1/20000 dilution + HeLa cell lysate at 10 µg

Secondary
Goat anti-rabbit HRP labeled at 1/2000 dilution

Predicted band size: 40 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?

Overlay histogram showing HeLa cells stained with ab51132 (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 (ab51132, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Ab51132 (1:100) staining human CAB39/MO25 in human brain tissue by immunohistochemistry using paraffin embedded tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunofluorescent staining of HeLa cells using ab51132 (1:100).