Lanes 1 - 4: Merged signal (red and green). Green - ab105217 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab105217 was shown to recognize in wild-type HAP1 cells as signal was lost at the expected MW in MGEA5 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and MGEA5 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab105217 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab105217 stained SKNSH cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab105217 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.