Recombinant Anti-ATAD2 antibody [EPR12730] (ab176319)

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: ATAD2 knockout HAP1 whole cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab176319 observed at 200 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab176319 was shown to specifically react with ATAD2 in wild type cells as signal was lost in ATAD2 knockout cells. Wild-type and ATAD2 knockout samples were subjected to SDS-PAGE. Ab176319 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 500 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.

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling ATAD2 (red) with ab176319 at a 1/60 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies. 

Immunofluorescent staining of HeLa cells labeling ATAD2 using ab176319 at a 1/100 dilution.

Lanes 1- 2: Merged signal (red and green). Green - ab176319 observed at 200 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab176319 was shown to react with ATAD2 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab264957 (CRISPR/Cas9 edited cell lysate ab257359) lane below 200kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and ATAD2 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab176319 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.