Immunocytochemistry/ Immunofluorescence analysis of PP2C alpha/PPM1A in HeLa cells. The cell was stained with ab14824 (1:100). The secondary antibody (green) was used Alexa Fluor 488. DAPI was stained the cell nucleus (blue).

All lanes : Anti-PPM1A antibody [p6c7] (ab14824) at 1/500 dilution

Lane 1 : Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : PPM1A knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : HAP1 whole cell lyate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole 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: 42 kDa
Observed band size: 42 kDa

Lanes 1-4: Merged signal (red and green). Green - ab14824 observed at 42 kDa. Red - loading control ab181602 observed at 36 kDa.

 ab14824 Anti-PPM1A antibody [p6c7]  was shown to specifically react with PPM1A in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265348 (knockout cell lysate ab259055) was used. Wild-type and PPM1A knockout samples were subjected to SDS-PAGE. ab14824 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 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.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PPM1A knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab14824 observed at 42 kDa. Red - loading control, ab18251, observed at 52 kDa.

ab14824 was shown to specifically react with PPM1A when PPM1A knockout samples were used. Wild-type and PPM1A knockout samples were subjected to SDS-PAGE. ab14824 diluted to 1/250 and ab18251 (loading control to alpha Tubulin) diluted to 1/10000 were incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

This image was generated using the ascites version of the product.

All lanes : Anti-PPM1A antibody [p6c7] (ab14824) at 1/1000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : K-562 cell lysate
Lane 4 : MCF7 cell lysate
Lane 5 : A549 cell lysate
Lane 6 : Raji cell lysate
Lane 7 : Mouse kidney tissue lysate
Lane 8 : Mouse brain tissue lysate
Lane 9 : Mouse liver tissue lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : goat anti-mouse secondary antibody conjugated to HRP

Predicted band size: 42 kDa

Western blot analysis of mouse liver cytosol extract using ab14824 at a dilution of 1/250.  Proteins were visualised using a goat anti-mouse secondary antibody conjugated to HRP and a DAB detection system. Western blot analysis of mouse liver cytosol extract using ab14824 at a dilution of 1/250. Proteins were visualised using a goat anti-mouse secondary antibody conjugated to HRP and a DAB detection system.

This image was generated using the ascites version of the product.

Anti-PPM1A antibody [p6c7] (ab14824) at 1/1000 dilution + HeLa whole cell lysate

Secondary
HRP conjugated anti-mouse antibody

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 42 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?


Exposure time: 10 seconds

This image is courtesy of an Abreview submitted by Xia Lin on 2 March 2006.

This image was generated using the ascites version of the product.

See Abreview

Ab14824 staining human colon. Staining is localised to cytoplasm.
Left panel: with primary antibody at 4ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

This image was generated using the ascites version of the product.