Lane 1: NIH3T3 whole cell lysate (10 µg)
Lane 2: Mouse brain whole tissue lysate (10 µg)
Lane 3: HeLa whole cell lysate (10 µg)
Lane 4: Hap1 Wild-type whole cell lysate (10 µg)
Lane 5: CANX knockout Hap1 whole cell lysate (10 µg)

Lanes 1 - 5: Merged signal (red and green). Green - ab213243 observed at 75 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab213243 was shown to specifically react with Calnexin in wild-type HAP1 cells as signal was lost in Calnexin (CANX) knockout cells. Wild-type and CANX knockout samples were subjected to SDS-PAGE. Nitrocellulose membranes were blocked in 3% milk in TBST before ab213243 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1µg/mL and 1/10,000 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 1/10,000 dilution for 1 hour at room temperature before imaging.

ab213243 staining Calnexin in NIH3T3 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 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 ab213243 at 0.05ugml then detected with an Alexa Fluor^® 488 goat anti-rabbit secondary antibody (ab150081) at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red).

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

ab213243 staining Calnexin in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 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 ab213243 at 0.05ugml then detected with an Alexa Fluor^® 488 goat anti-rabbit secondary antibody (ab150081) at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red).

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