All lanes : Anti-PP-T antibody [12F7] (ab223367) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : PPP5C knockout HEK293T cell lysate
Lane 3 : A431 cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Predicted band size: 56 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab223367 observed at 58 kDa. Red - loading control ab181602 observed at 36 kDa.

 ab223367 Anti-PP-T antibody [12F7] was shown to specifically react with PP-T in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266507 (knockout cell lysate ab258136) was used. Wild-type and PP-T knockout samples were subjected to SDS-PAGE. ab223367 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

4% formaldehyde-fixed HeLa (human epithelial cell line from cervix adnocarcinoma) cells stained for PP-T (green) using ab223367 at 1/100 dilution for 60 minutes at room temperature in ICC/IF, followed by Goat Anti-Mouse ATTO 488 at 1/100 dilution for 60 minutes at room temperature. Counterstain: DAPI (blue) nuclear stain at 1/5000 for 5 minutes room temperature.

(A) DAPI nuclear stain (B) ab223367 (C) Merge.

All lanes : Anti-PP-T antibody [12F7] (ab223367) at 1/500 dilution

Lane 1 : A431 (human epidermoid carcinoma cell line) cell lysate
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) cell lysate
Lane 3 : Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG HRP at 1/200 dilution

Developed using the ECL technique.

Predicted band size: 56 kDa
Observed band size: 58 kDa why is the actual band size different from the predicted?


Exposure time: 6 minutes

Blocking buffer: 5% skim milk.

Primary and secondary antibodies were incubated for 1 hour at room temperature.

2% formaldehyde-fixed HEK-293 (human epithelial cell line from embryonic kidney) cells stained for PP-T (green) using ab223367 at 1/50 dilution in ICC/IF, followed by Goat Anti-Mouse Alexa Fluor 488 at 1/100 dilution for 60 minutes at RT. Counterstain: DAPI (blue) nuclear stain.

(A) DAPI nuclear stain (B) ab223367 (C) Merge.