All lanes : Anti-PDK1 antibody [EPR13009] (ab175190) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PDK1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 49 kDa
Observed band size: 46 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab175190 observed at 46 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab175190 was shown to react with PDK1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264791 (knockout cell lysate ab257164) was used. Wild-type HeLa and PDK1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab175190 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-PDK1 antibody [EPR13009] (ab175190) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : PDK1 knockout HAP1 cell lysate
Lane 3 : Human fetal heart lysate
Lane 4 : LnCaP cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 49 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab175190 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab175190 was shown to specifically recognize PDK1 in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when PDK1 knockout samples were examined. Wild-type and PDK1 knockout samples were subjected to SDS-PAGE. ab175190 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 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 hour at room temperature before imaging.

All lanes : Anti-PDK1 antibody [EPR13009] (ab175190) at 1/1000 dilution

Lane 1 : LNCaP lysates
Lane 2 : Human fetal heart lysates

Lysates/proteins at 10 µg per lane.

Predicted band size: 49 kDa

Western blot analysis on immunoprecipitation pellet from Human fetal heart lysate (lane 1) labeling PDK1 with ab175190 at 1/10 dilution or 1X PBS (lane 2) negative control.  HRP-conjugated anti-Rabbit IgG preferentially detecting the non-reduced form of Rabbit IgG.