Anti-F-actin antibody [4E3.adl] - Loading Control

• WB

Project

Western blot - Anti-F-actin antibody [4E3.adl] - Loading Control (AB130935)

All lanes:

Western blot - Anti-F-actin antibody [4E3.adl] - Loading Control (ab130935) at 1/500 dilution

Lane 1:

Human skeletal muscle tissue lysate - total protein (<a href='/en-us/products/unavailable/human-skeletal-muscle-tissue-lysate-total-protein-ab29330'>ab29330</a>) at 20 µg

Lane 2:

Skeletal Muscle (Mouse) Tissue Lysate at 20 µg

Lane 3:

Heart (Mouse) Tissue Lysate at 20 µg

Lane 4:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 20 µg

Lane 5:

C2C12 (Mouse myoblast cell line) Whole Cell Lysate at 20 µg

Secondary

All lanes:

Peroxidase- conjugated AffiniPure Goat Anti-mouse IgM (ab98112) at 1/3000 dilution

Predicted band size: 41 kDa

Observed band size: 42 kDa,60 kDa,70 kDa

true

Exposure time: 10s

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-F-actin antibody [4E3.adl] - Loading Control (AB130935)

ab130935 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 ab130935 at 5μg/ml overnight at +4°C. The secondary antibody (green) was a goat anti-mouse DyLight® 488 (ab96879) IgG (H+L) used at a 1/250 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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-F-actin antibody [4E3.adl] - Loading Control (AB130935)

Overlay histogram showing HeLa cells stained with ab130935 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 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 (ab130935, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was a goat Anti-mouse DyLight^® 488 (IgM; mu chain, ab97007) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgM [ICIGM] (ab91545, 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. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Triton X-100 for 20 min used under the same conditions.