Lanes 1, 3 and 5: Wild-type HAP1 cell lysate (20 µg)
Lanes 2, 4 and 6: CDK4 knockout HAP1 cell lysate (20 µg)
Lanes 1 and 2: Green signal from target - ab108355 observed at 34 kDa
Lanes 3 and 4: Red signal from loading control - ab8226 observed at 42 kDa
Lanes 5 and 6: Merged (red and green) signal
ab108355 was shown to specifically react with CDK4 when CDK4 knockout samples were used. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. ab108355 and ab8226 (loading control to beta actin) were both diluted at 1/1000 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.

ab108355 staining Cdk4 in wild-type HAP1 cells (top panel) and Cdk4 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab108355 at 1/400 dilution and ab7291 at 1μg/ml concentration 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) and a goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150120) (shown in pseudo colour red). Nuclear DNA was labelled in blue with DAPI.

Immunofluorescence staining of MCF7 cells with purified ab108355 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab108355 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

All lanes : Anti-Cdk4 antibody [EPR4513-54-3] (ab108355) at 1/5000 dilution (purified)

Lane 1 : HeLa cell lysate
Lane 2 : K562 cell lysate
Lane 3 : Ramos cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/50000 dilution

Predicted band size: 34 kDa
Observed band size: 34 kDa

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

All lanes : Anti-Cdk4 antibody [EPR4513-54-3] (ab108355) at 1/1000 dilution (unpurified)

Lane 1 : Human osteosarcoma cells - control, non-targeting siRNA
Lane 2 : Human osteosarcoma cells - siRNA for CDK4

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG polyclonal at 1/2000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 34 kDa
Observed band size: 34 kDa


Exposure time: 5 seconds

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