Recombinant Anti-PDCD4 antibody [EPR3431] (ab80590)

Lanes 1- 2: Merged signal (red and green). Green - ab80590 observed at 51 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab80590 was shown to react with PDCD4 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261833 (knockout cell lysate ab257278) was used. Wild-type HeLa and PDCD4 knockout 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. ab80590 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 20000 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.

Lanes 1 - 4: Merged signal (red and green). Green - ab80590 observed at 52 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab80590 was shown to specifically react with PDCD4 in wild-type HAP1 cells as signal was lost in PDCD4 knockout cells. Wild-type and PDCD4 knockout samples were subjected to SDS-PAGE. ab80590 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C both at 1/20000 dilution. 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 1/10000 dilution for 1 hour at room temperature before imaging.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PDCD4 with purified ab80590 at 1/1000 dilution (1 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).

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate, 10µg
Lane 2 (+): ab80590 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab80590 in HeLa whole cell lysate

Ab80590 (Purified) at 1/30 dilution (20 µg/ml) immunoprecipitating PDCD4 in HeLa whole cell lysate. For western blotting, ab80590 at 1/500 and VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM /TBST .