Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: TARDBP knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: Jurkat whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - unpurified ab133547 observed at 44 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab133547 was shown to specifically react with TARDBP in wild-type HAP1 cells. No band was observed when TARDBP knockout samples were examined. Wild-type and TARDBP knockout samples were subjected to SDS-PAGE. Ab133547 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10,000 dilution 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebrum tissue sections labeling TDP43 with purified ab133547 at 1/350 dilution (1.92 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling TDP43 with purified ab133547 at 1/100 dilution (10 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% TritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling TDP43 with purified ab133547 at 1/70 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebrum tissue sections labeling TDP43 with purified ab133547 at 1/350 dilution (1.92 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Unpurfied ab133547 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 unpurified ab133547 at 1 μg/mL and ab195889 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 Rabbit IgG (Alexa Fluor® 488) (ab150081) 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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebrum tissue sections labeling TDP43 with purified ab133547 at 1/350 dilution (1.92 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-TDP43 antibody [EPR5811] (ab133547) at 1/1000 dilution (purified)

Lane 1 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 2 : Human brain lysate
Lane 3 : Mouse brain lysate
Lane 4 : Rat brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 44 kDa
Observed band size: 44 kDa

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling TARDBP with unpurified ab133547 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.