Anti-Filamin A antibody [EP2405Y]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Filamin A antibody [EP2405Y] (AB76289)

Immunofluorescent staining of HeLa cells using ab76289 at a 1/100 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Filamin A antibody [EP2405Y] (AB76289)

ab76289 staining Filamin Ain the human cell line HeLa (human cervix adenocarcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Filamin A antibody [EP2405Y] (AB76289)

Overlay histogram showing A431 cells stained with ab76289 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton 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 (ab76289 , 1/100 dilutio) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in A431 cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Triton used under the same conditions.

• WB

Lab

Western blot - Anti-Filamin A antibody [EP2405Y] (AB76289)

Blocking buffer : 5% NFDM/TBST

Diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Filamin A antibody [EP2405Y] (ab76289) at 1/10000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

COS-1 (Cercopithecus aethiops kidney) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), HRP conjugated at 1/1000 dilution

Predicted band size: 281 kDa

Observed band size: 281 kDa

false

Exposure time: 30s

• WB

Lab

Western blot - Anti-Filamin A antibody [EP2405Y] (AB76289)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : FLNA knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HepG2 whole cell lysate (20 μg)
Lane 4 : HeLa whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab76289 observed at 281 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab76289 was shown to specifically react with FLNA when FLNA knockout samples were used. Wild-type and FLNA knockout samples were subjected to SDS-PAGE. ab76289 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 250000 dilution and 1/10000 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-Filamin A antibody [EP2405Y] (ab76289)

Predicted band size: 281 kDa

false

• WB

Unknown

Western blot - Anti-Filamin A antibody [EP2405Y] (AB76289)

All lanes:

Western blot - Anti-Filamin A antibody [EP2405Y] (ab76289) at 1/500000 dilution

Lane 1:

COS-1 (African green monkey kidney fibroblast-like cell line) cell lysate at 10 µg

Lane 2:

Hela cell lysate at 10 µg

Lane 3:

3T3 cell lysate at 10 µg

Lane 4:

C6 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/1000 dilution

Predicted band size: 281 kDa

Observed band size: 281 kDa

false