All lanes : Anti-PTEN antibody [EPR4408-76] (ab133532) at 1/10000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 47 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab133532 observed at 47 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

 ab133532 was shown to react with PTEN in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255419 (knockout cell lysate ab263829) was used. Wild-type HeLa and PTEN 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. ab133532 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 10000 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-PTEN antibody [EPR4408-76] (ab133532) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PTEN knockout HeLa cell lysate
Lane 3 : HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 47 kDa
Observed band size: 47 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab133532 observed at 47 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab133532 was shown to react with PTEN in wild-type HeLa cells in western blot. The bands observed in PTEN knockout cell line ab255419 (PTEN knockout cell lysate ab263829) below 47 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. HeLa wild-type and PTEN knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab133532 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

Lanes 1-2 : Anti-PTEN antibody [EPR4408-76] (ab133532) at 1/10000 dilution
Lanes 3-4 : Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/2000 dilution


Lanes 1 & 3 & 5 : PTEN knockout HAP1 cell lysate
Lanes 2 & 4 & 6 : Wild-type HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 47 kDa

Lanes 1 and 2: Green signal from target - ab133532 observed at 47 kDa
Lanes 3 and 4: Red signal from loading control - ab8245 observed at 37 kDa
Lanes 5 and 6: Merged (red and green) signal

ab133532 was shown to specifically react with PTEN in wild-type HAP1 cells. No band was observed when PTEN knockout samples were used. Wild-type and PTEN knockout samples were subjected to SDS-PAGE, ab133532 and ab8245 (loading control to GAPDH) were diluted to 1/10,000 and 1/2000 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 1hr at room temperature before imaging.

All lanes : Anti-PTEN antibody [EPR4408-76] (ab133532) at 1/10000 dilution

Lane 1 : C6 cell lysate
Lane 2 : RAW 264.7 cell lysate
Lane 3 : NIH 3T3 cell lysate
Lane 4 : MCF7 cell lysate
Lane 5 : 293T cell lysate
Lane 6 : A431 cell lysate
Lane 7 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 47 kDa
Observed band size: 54 kDa why is the actual band size different from the predicted?