All lanes : Anti-PDK4 antibody [1C2BG5] (ab110336) at 1/500 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PDK4 knockout HeLa cell lysate
Lane 3 : Human heart tissue lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 46 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab110336 observed at 48 kDa. Red - loading control, ab129002 observed at 124 kDa.

ab110336 Anti-PDK4 antibody [1C2BG5] was shown to specifically react with PDK4 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261805 (knockout cell lysate ab257217) was used. Wild-type and PDK4 knockout samples were subjected to SDS-PAGE. ab110336 and Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 1000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

ICC/IF image of ab110336 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab110336, 10µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Overlay histogram showing HepG2 cells stained with ab110336 (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. The cells were then incubated with the antibody (ab110336, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

All lanes : Anti-PDK4 antibody [1C2BG5] (ab110336) at 1 µg/ml

Lane 1 : HepG2 whole cell lysate at 10 µg
Lane 2 : Human heart tissue at 10 µg
Lane 3 : Recombinant Human PDK1 at 0.01 µg
Lane 4 : Recombinant Human PDK2 at 0.01 µg
Lane 5 : Recombinant Human PDK3 at 0.01 µg
Lane 6 : Recombinant Human PDK4 at 0.01 µg

Predicted band size: 46 kDa