Western blot - Anti-Cdk6 antibody [EPR4515] (ab124821)
Predicted band size : 37 kDa
Lanes 1, 3 and 5: Wild-type HAP1 cell lysate (20 µg) Lanes 2, 4 and 6: CDK6 knockout HAP1 cell lysate (20 µg) Lanes 1 and 2: Green signal from target - ab124821 observed at 37 kDa Lanes 3 and 4: Red signal from loading control - ab8226 observed at 42 kDa Lanes 5 and 6: Merged (red and green) signal ab124821 was shown to specifically react with CDK6 when CDK6 knockout samples were used. Wild-type and CDK6 knockout samples were subjected to SDS-PAGE. ab124821 and ab8226 (loading control to beta actin) were diluted at 1/10 000 and 1/1000 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 1/10,000 dilution for 1 h at room temperature before imaging.
ab124821 staining Cdk6 in wild-type HAP1 cells (top panel) and Cdk6 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 ab124821 at 1/500 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Overlay histogram showing HeLa cells stained with ab124821 (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 (ab124821, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/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.
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