Anti-PDCD4 antibody [EPR3432]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDCD4 antibody [EPR3432] (AB79405)

ab79405 at 1/1000 dilution staining PDCD4 in human breast carcinoma by Immunohistochemistry using paraffin-embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PDCD4 antibody [EPR3432] (AB79405)

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.

• WB

Lab

Western blot - Anti-PDCD4 antibody [EPR3432] (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.

All lanes:

Western blot - Anti-PDCD4 antibody [EPR3432] (ab79405) at 1/5000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PDCD4 knockout HeLa cell lysate at 20 µg

Predicted band size: 51 kDa

Observed band size: 51 kDa

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• WB

Lab

Western blot - Anti-PDCD4 antibody [EPR3432] (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.

All lanes:

Western blot - Anti-PDCD4 antibody [EPR3432] (ab79405) at 1/5000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

PDCD4 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

HEK293 whole cell lysate at 20 µg

Predicted band size: 51 kDa

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• WB

Unknown

Western blot - Anti-PDCD4 antibody [EPR3432] (AB79405)

All lanes:

Western blot - Anti-PDCD4 antibody [EPR3432] (ab79405) at 1/10000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 51 kDa

Observed band size: 30 kDa,52 kDa

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