All lanes : Anti-PPP2R5E antibody [EPR17147] - C-terminal (ab198500) at 1/5000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PPP2R5E knockout HeLa cell lysate
Lane 3 : HEK-293 cell lysate
Lane 4 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 55 kDa

Lanes 1-4: Merged signal (red and green). Green - ab198500 observed at 55 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab198500 Recombinant Anti-PPP2R5E antibody [EPR17147] - C-terminal was shown to specifically react with PPP2R5E in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265637 (knockout cell lysate ab258135) was used. Wild-type and PPP2R5E knockout samples were subjected to SDS-PAGE. ab198500 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 dilution and 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.

 

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling PPP2R5E with ab198500 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on Human cervix carcinoma tissue isobserved. Counter-stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF-7 (Human breast carcinoma) cells labeling PPP2R5E with ab198500 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Cytoplasm staining on MCF7 cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
1. ab198500 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

 

Flow cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PPP2R5E (red) with purified ab198500 at a 1/2500 dilution (1ug/mL). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).

All lanes : Anti-PPP2R5E antibody [EPR17147] - C-terminal (ab198500) at 1/5000 dilution

Lane 1 : HeLa cell lysate at 10 µg
Lane 2 : 293 cell lysate at 10 µg
Lane 3 : Human skeletal muscle lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 55 kDa
Observed band size: 55 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

PPP2R5E was immunoprecipitated from 1mg of HeLa (Human cervix adenocarcinoma) whole cell extract with ab198500 at 1/175 dilution. Western blot was performed from the immunoprecipitate using ab198500 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: HeLa whole cell extract 10µg (Input).

Lane 2: HeLa whole cell extract

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab198500 in HeLa whole cell extract.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human cervix adenocarcinoma) cells labeling PPP2R5E with ab198500 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Cytoplasm staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
1. ab198500 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

 

Immunohistochemical analysis of paraffin-embedded Human transitional cell carcinoma of bladder tissue labeling PPP2R5E with ab198500 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasm staining on Human transitional cell carcinoma of bladder tissue isobserved. Counter-stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.