Recombinant Anti-Integrin linked ILK antibody [EPR1592] (ab76468)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Integrin linked ILK knockout HAP1 cell lysate (20 µg)
Lane 3: Hek293 cell lysate (20 µg)
Lane 4: K562 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab76468 observed at 51 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab76468 was shown to specifically react with Integrin linked ILK when Integrin linked ILK knockout samples were used. Wild-type and Integrin linked ILK knockout samples were subjected to SDS-PAGE. ab76468 and ab8245 (loading control to GAPDH) were diluted at 1/5000 and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.

Immunocytochemistry/Immunofluorescence analysis of A431 (human epidermoid carcinoma) cells labelling Integrin linked ILK (green) with purified ab76468 at 1/500. Cells were fixed with 100% methanol. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were stained blue with DAPI.  

Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

Overlay histogram showing HEK293 cells stained with ab76468 (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 (ab76468, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit DyLight® 488 (IgG; H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/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 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.