Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: non-muscle Myosin IIA knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: HEK293 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab55456 observed at 230 kDa. Red - loading control, ab18251, observed at 52 kDa.

ab55456 was shown to specifically react with non-muscle Myosin IIA when non-muscle Myosin IIA knockout samples were used. Wild-type and non-muscle Myosin IIA knockout samples were subjected to SDS-PAGE. ab55456 at a concentration of 1 µg/ml and ab18251 (loading control to alpha Tubulin) at a dilution of 1/1000 were incubated overnight at 4°C. Blots were developed withGoat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Immunofluorecence analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling non-muscle Myosin IIA (Green) using ab55456 at 10 µg/ml in ICC/IF analysis.

Immunofluorescence analysis of plakoglobin-knocked down MCF7 cells, staining non-muscle Myosin IIA with ab55456.

Cells were fixed with formaldehyde, permeabilized with 0.2% Triton X-100 and blocked in 5% goat serum. Samples were incubated with primary antibody overnight at 4°C. An AlexaFluor®-conjugated anti-mouse IgG was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling non-muscle Myosin llA with ab55456 at 0.5 µg/ml.

Overlay histogram showing HEK293 cells stained with ab55456 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab55456, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 100% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

ICC/IF image of ab55456 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab55456, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Western blot against tagged recombinant protein immunogen using ab55456 non-muscle Myosin IIA antibody at 1ug/ml. Predicted band size of immunogen is 33 kDa

Anti-non-muscle Myosin IIA antibody [2B3] (ab55456) + HeLa

Anti-non-muscle Myosin IIA antibody [2B3] (ab55456) at 5 µg/ml + Human kidney tissue lysate