Recombinant Anti-DDX5 antibody [EPR7239] (ab126730)

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: DDX5 knockout HAP1 whole cell lysate (20 µg)
Lane 3: MCF7 whole cell lysate (20 µg)
Lane 4: HeLa whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab126730 observed at 69 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab126730 was shown to specifically react with DDX5 in wild-type cells as signal was lost in DDX5 knockout cells. Wild-type and DDX5 knockout samples were subjected to SDS-PAGE. Ab126730 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution 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.

ab126730 staining DDX5 in the human cell line HeLa (Human cervix adenocarcinoma epithelial cell) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilised with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/200. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody. 

Isoytype control: Rabbit monoclonal IgG (Black) Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue). 

Immunocytochemistry/Immunofluorescence analysis of SH-SY5Y (human neuroblastoma) cells labelling DDX5 (green) with purified ab126730 at 1/250. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as a nuclear counterstain.  

Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

ab126730, at a dilution of 1/250, staining DDX5 in paraffin-embedded Human bladder carcinoma tissue by Immunohistochemistry.

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

Purified ab126730 at 1/20 dilution (1µg) immunoprecipitating DDX5 in Mouse brain lysate.
Lane 1 (input): Mouse brain lysate 10µg
Lane 2 (+): ab126730 + Mouse brain lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab126730 in Mouse brain lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/20,000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 69 kDa

ab126730, at a dilution of 1/250, staining DDX5 in paraffin-embedded Human kidney tissue by Immunohistochemistry.

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