All lanes : Anti-PDCD4 antibody [EPR3432] (ab79405) at 1/5000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PDCD4 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 52 kDa
Observed band size: 51 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab79405).

  Lanes 1- 2: Merged signal (red and green). Green - ab79405 observed at 51 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab79405 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. ab79405 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 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.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling PDCD4 with purified ab79405 at 1/500 dilution (4 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. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL) was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab79405).

All lanes : Anti-PDCD4 antibody [EPR3432] (ab79405) at 1/5000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PDCD4 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HEK293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 52 kDa

This data was developed using the same antibody clone in a different buffer formulation (ab79405).

Lanes 1 - 4: Merged signal (red and green). Green - ab79405 observed at 52 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab79405 was shown to recognize PDCD4 in wild-type HAP1 cells as signal was lost at the expected MW in PDCD4 knockout HAP1 cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and PDCD4 knockout samples were subjected to SDS-PAGE. ab79405 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 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 1/10000 dilution for 1 hour at room temperature before imaging.