All lanes : Anti-MCPyV_gp3 large T antigen antibody [Ab3] (ab202866) at 0.5 µg/ml

Lane 1 : MKL-1 whole cell lysate
Lane 2 : HeLa whole cell lysate
Lane 3 : HEK293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 48 kDa why is the actual band size different from the predicted?

This blot was produced using a 4-12% Bis-tris under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was blocked for an hour using 5% milk before ab202866 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at a 0.5ug/ml concentration and 1/20000 dilution respectively. Antibody binding was detected using Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab202866 staining MCPyV_gp3 large T antigen in MKL-1 cells. The cells were fixed with 4% PFA (10min), permeabilized with 0.1%PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab202866 at 5μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labelled with DAPI (shown in blue).

 

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).