All lanes : Anti-PDCD4 antibody [EPR3431] (ab80590) at 1/20000 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?

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.

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma epithelial cell) labelling with ab80590 at a dilution of 1:100 dilution (0.34 µg/ml). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) (1:1000 dilution (2 µg/ml)) was used as the secondary antibody. The cells were co-stained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/ml). Nuclei counterstained with DAPI (blue). Control: PBS instead of the primary antibody.

All lanes : Anti-PDCD4 antibody [EPR3431] (ab80590) at 1/20000 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

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 : Anti-PDCD4 antibody [EPR3431] (ab80590) at 1/10000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : HL60 + RA lysate
Lane 3 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 52 kDa
Observed band size: 52 kDa

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 .

Immunohistochemistry analysis of paraffin-embedded Human tonsil tissue using 1/100 ab80590.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Equilibrium disassociation constant (K_D)
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