All lanes : Anti-PKC zeta antibody [EP1490(2)] (ab108970) at 1/1000 dilution

Lane 1 : 293T lysate
Lane 2 : HT-29 lysate
Lane 3 : Fetal liver lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 74 kDa
Observed band size: 74 kDa

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling PKC zeta with unpurified ab108970 at 1/150 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti-rabbit IgG (Alexa Fluor^® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PKC zeta knockout HAP1 cell lysate (20 µg)
Lane 3: HT-29 cell lysate (20 µg)
Lane 4: A431 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab108970 observed at 74 kDa. Red - loading control, ab8226, observed at 42 kDa.
ab108970 was shown to specifically react with PKC zeta when PKC zeta knockout samples were used. Wild-type and PKC zeta knockout samples were subjected to SDS-PAGE. ab108970 and ab8226 (loading control to beta actin) were both diluted 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 1/10000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-PKC zeta antibody [EP1490(2)] (ab108970) at 1/1000 dilution

Lane 1 : A431 whole cell lysate
Lane 2 : Mouse lung tissue lysate
Lane 3 : Rat lung tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 74 kDa
Observed band size: 74 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab108970 observed at 74 kDa. Red - loading control, ab8245, observed at 37 kDa.

 

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab108970 and ab8245 (loading control) overnight at 4°C. Antibody binding was detected using Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at a 1:10000 dilution for 1hr at room temperature and then imaged.