Anti-TDP43 antibody [3H8]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [3H8] (AB104223)

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).

• Flow Cyt

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Flow Cytometry - Anti-TDP43 antibody [3H8] (AB104223)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [3H8] (AB104223)

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).

• WB

Lab

Western blot - Anti-TDP43 antibody [3H8] (AB104223)

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.

All lanes:

Western blot - Anti-TDP43 antibody [3H8] (ab104223)

Predicted band size: 44 kDa

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