All lanes : Anti-alpha Actinin/ACTN1 antibody [MAC 276] (ab50599) at 1 µg/ml

Lane 1 : Human skeletal muscle tissue lysate - total protein (ab29330)
Lane 2 : Heart (Rat) Tissue Lysate
Lane 3 : Skeletal Muscle (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase Conjugated AffiniPure Rabbit Anti-Rat IgG (H+L) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 103 kDa
Observed band size: 103 kDa


Exposure time: 8 minutes

IHC image of alpha Actin staining in Human normal bladder formalin fixed paraffin embedded tissue section. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 20 mins at 98°C. The section was incubated with ab50599, 5µg/ml, for 15 mins at room temperature. A Goat anti-Rat biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. The section was counterstained with haematoxylin and mounted with DPX.

ICC/IF image of ab68826 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 ab50599 5µg/ml overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti- rat 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.

Overlay histogram showing HeLa cells stained with ab50599 (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 (ab50599, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rat IgG2a, kappa monoclonal [aRTK2758] (ab18450, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.