All lanes :

Lane 1 : HAP1 whole cell lysate
Lane 2 : HAP1 Src KO whole cell lysate
Lane 3 : A431 whole cell lysate
Lane 4 : Mouse testes whole tissue lysate
Lane 5 : Rat testes whole tissue lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 60 kDa

ab231081 was shown to specifically react with Src in wild type HAP1 cells. No band was observed when Src knockout samples were used. Wild-type and Src knockout samples were subjected to SDS-PAGE. ab231081 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at a 1ug/ml concentration and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab231081 staining Src in wild-type HAP1 cells (top panel) and SRC knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (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 ab231081 at 5μg/ml and ab6046 (Rabbit polyclonal to beta tubulin - loading control) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with ab150117 (Goat secondary antibody to Mouse IgG (Alexa Fluor® 488) at 2 μg/ml (colored green) and ab150084 (Goat secondary antibody to Rabbit IgG (Alexa Fluor® 594) at 2 μg/ml (pseudo color red). Nuclear DNA was labelled in blue with DAPI.

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