Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: TDP43 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab104223 observed at 48 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab104223 was shown to specifically react with TDP43 when TDP43 knockout samples were used. Wild-type and TDP43 knockout samples were subjected to SDS-PAGE.  Ab104223 and ab181602 (loading control to GAPDH) were diluted at 1/5000 and 1/10,000 dilution respectively and incubated overnight at 4C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

ab104223 staining TDP43 in wild-type HAP1 cells (top panel) and TARDBP knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 with ab104223 at 1/500 dilution and ab202272 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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

ab104223 at 1/1000 dilution, staining TARDBP in rat brain tissue (red). Chicken antibody to GFAP CPCA-GFAP (green) shows the processes of astrocytic glial cells. Nuclei of all cells are revealed with DAPI DNA stain (blue).

Overlay histogram showing JEG3 cells stained with ab104223 (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 (ab104223, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 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. This antibody gave a positive signal in JEG3 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.