ab108198 staining ACTN4 in wild-type HAP1 cells (top panel) and ACTN4 knockout HAP1 cells (bottom panel). The cells were fixed with PFA (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab108198 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: ACTN4 (alpha Actinin 4) knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: MCF7 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab108198 observed at 105 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab108198 was shown to specifically react with alpha Actinin 4 in wild-type HAP1 cells as signal was lost in ACTN4 (alpha Actinin 4) knockout cells. Wild-type and ACTN4 (alpha Actinin 4) knockout samples were subjected to SDS-PAGE. ab108198 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.

ab108198 staining alpha Actinin 4 in the human cell line HeLa (human cervix adenocarcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. The sample was incubated with the primary antibody at a dilution of 1/50. 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)

Purified ab108198 at 1/50 dilution (2µg) immunoprecipitating alpha Actinin 4 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab108198 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab108198 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 105 kDa

All lanes : Anti-alpha Actinin 4 antibody [EPR2533(2)] (ab108198) at 1/1000 dilution

Lane 1 : Fetal skeletal muscle lysate
Lane 2 : A431 cell lysate
Lane 3 : MCF-7 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 105 kDa

ab108198, at 1/100 dilution, staining alpha Actinin 4 in paraffin-embedded Human kidney tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab108198, at 1/100 dilution, staining alpha Actinin 4 in HeLa cells by Immunofluorescence.