Anti-Fascin antibody [EP5902]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fascin antibody [EP5902] (AB126772)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Fascin antibody [EP5902] (AB126772)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fascin antibody [EP5902] (AB126772)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fascin antibody [EP5902] (AB126772)

ab126772 showing positive staining in Normal tonsil tissue.

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Fascin antibody [EP5902] (AB126772)

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.

• IP

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Immunoprecipitation - Anti-Fascin antibody [EP5902] (AB126772)

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:

Immunoprecipitation - Anti-Fascin antibody [EP5902] (ab126772)

Predicted band size: 54 kDa

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• WB

Lab

Western blot - Anti-Fascin antibody [EP5902] (AB126772)

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:

Western blot - Anti-Fascin antibody [EP5902] (ab126772) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

FSCN1 (Fascin) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HepG2 whole cell lysate at 20 µg

Predicted band size: 54 kDa

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• WB

Lab

Western blot - Anti-Fascin antibody [EP5902] (AB126772)

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.

All lanes:

Western blot - Anti-Fascin antibody [EP5902] (ab126772) at 1/1000 dilution

Lane 1:

Wild-type HCT116 cell lysate at 20 µg

Lane 2:

FSCN1 knockout HCT116 cell lysate at 20 µg

Lane 2:

Western blot - Human FSCN1 (Fascin) knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-fscn1-fascin-knockout-hct116-cell-line-ab266895'>ab266895</a>)

Lane 3:

SH-SY5Y cell lysate at 20 µg

Lane 4:

K-562 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 54 kDa

Observed band size: 60 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fascin antibody [EP5902] (AB126772)

ab126772 showing positive staining in Normal kidney tissue.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fascin antibody [EP5902] (AB126772)

ab126772 showing positive staining in Glioma tissue.

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

• WB

Unknown

Western blot - Anti-Fascin antibody [EP5902] (AB126772)

All lanes:

Western blot - Anti-Fascin antibody [EP5902] (ab126772) at 1/10000 dilution

Lane 1:

SH SY5Y cell lysate at 10 µg

Lane 2:

K562 cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit at 1/2000 dilution

Predicted band size: 54 kDa

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