Anti-PPM1A 抗体 [p6c7] (ab14824)

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).

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.

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.

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.

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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.