Lanes 1-2 : Anti-Talin 1 antibody [8D4] (ab157808) at 1/1000 dilution
Lanes 3-4 : Anti-Talin 1 (phospho S425) antibody (ab157807) at 1/1000 dilution (Rabbit polyclonal to anti-Talin 1 (phospho Serine 425))

Lanes 1 & 3 : PC12 cell lysate - stimulated with calyculin A at 100nM for 30 minutes
Lanes 2 & 4 : PC12 cell lysate - stimulated with calyculin A at 100nM for 30 minutes and treated with lambda phosphatase

Predicted band size: 269 kDa
Observed band size: 240 kDa why is the actual band size different from the predicted?



Western blot membranes were incubated with diluted antibody in 5% non-fat milk, PBS and 0.04% Tween20 for 1 hour at room temperature.

Overlay histogram showing RAW 264.7 cells stained with ab157808 (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 (ab157808, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 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 RAW 264.7 cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunocytochemical analysis of chick fibroblasts labelling Talin 1 with ab157808 at 1/100 (red). F-actin was stained in green.
Image provided by Dr. Gianluca Gallo at Drexel University.