All lanes : Anti-Fascin antibody [EP5902] (ab126772) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : FSCN1 knockout HCT116 cell lysate
Lane 3 : SH-SY5Y cell lysate
Lane 4 : K-562 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 54 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab126772 observed at 60 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab126772 Anti-Fascin antibody [EP5902] was shown to specifically react with Fascin in wild-type HCT116 cells. Loss of signal was observed when knockout cell line ab266895 (knockout cell lysate ab257444) was used. Wild-type and Fascin knockout samples were subjected to SDS-PAGE. ab126772 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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 U87-MG (human glioblastoma) labelling Fascin with purified ab126772 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) at dilution of 1/1000 was used as the secondary antibody. Nuclei couterstained with DAPI (blue). 

Secondary Only Control: PBS was used instead of the primary antibody as the negative control. 

ab126772, at 1/250 dilution, staining Fascin in paraffin-embedded Human Hodgkin's lymphoma tissue by Immunohistochemistry.

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

Overlay histogram showing HeLa cells stained with ab126772 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab126772, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Purified ab126772 at 1/20 dilution (0.5µg) immunoprecipitating Fascin in HepG2 whole cell lysate.
Lane 1 (input): HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab126772 + HepG2 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab126772 in HepG2 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/5000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 54 kDa

All lanes : Anti-Fascin antibody [EP5902] (ab126772) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : FSCN1 (Fascin) knockout HAP1 whole cell lysate
Lane 3 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 54 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab126772 observed at 54 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab126772 was shown to specifically react with Fascin in wild-type HAP1 cells as signal was lost in FSCN1 (Fascin) knockout cells. Wild-type and FSCN1 (Fascin) knockout samples were subjected to SDS-PAGE. Ab126772 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Fascin antibody [EP5902] (ab126772) at 1/10000 dilution

Lane 1 : SH SY5Y cell lysate
Lane 2 : K562 cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 54 kDa

ab126772, at 1/250 dilution, staining Fascin in paraffin-embedded Human colon tissue by Immunohistochemistry.

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

ab126772 showing positive staining in Normal tonsil tissue.

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

ab126772 showing positive staining in Glioma tissue.

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

ab126772 showing positive staining in Normal kidney tissue.

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