Anti-PDCD4 antibody [EPR3431]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDCD4 antibody [EPR3431] (AB80590)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue sections labeling PDCD4 with Purified ab80590 at 1 : 3000 dilution (0.05 µg/mL). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PDCD4 antibody [EPR3431] (AB80590)

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).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PDCD4 antibody [EPR3431] (AB80590)

Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PDCD4 with Purified ab80590 at 1 : 50 dilution (2.9 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. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDCD4 antibody [EPR3431] (AB80590)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast cancer tissue sections labeling PDCD4 with Purified ab80590 at 1 : 3000 dilution (0.05 µg/mL). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) . Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used. PBS instead of the primary antibody was used as the negative control.

• IP

Lab

Immunoprecipitation - Anti-PDCD4 antibody [EPR3431] (AB80590)

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 .

All lanes:

Immunoprecipitation - Anti-PDCD4 antibody [EPR3431] (ab80590)

Predicted band size: 51 kDa

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• WB

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Western blot - Anti-PDCD4 antibody [EPR3431] (AB80590)

All lanes:

Western blot - Anti-PDCD4 antibody [EPR3431] (ab80590) at 1/1000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 51 kDa

Observed band size: 52 kDa

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• WB

Lab

Western blot - Anti-PDCD4 antibody [EPR3431] (AB80590)

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.

All lanes:

Western blot - Anti-PDCD4 antibody [EPR3431] (ab80590) at 1/20000 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

Lab

Western blot - 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.

All lanes:

Western blot - Anti-PDCD4 antibody [EPR3431] (ab80590) at 1/20000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PDCD4 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PDCD4 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-pdcd4-knockout-hela-cell-line-ab261833'>ab261833</a>)

Predicted band size: 51 kDa

Observed band size: 51 kDa

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