All lanes : Anti-PP2A alpha + beta antibody [E155] (ab32104) at 1/5000 dilution

Lane 1 : Untreated HeLa (human cervix adenocarcinoma) whole cell lysates
Lane 2 : HeLa (human cervix adenocarcinoma) whole cell lysates,cells were treated with Pervanadate for 30 minutes at the final concentration of 1mg/ml
Lane 3 : HeLa (human cervix adenocarcinoma) whole cell lysates,cells were treated with Pervanadate for 30 minutes at the final concentration of 1mg/ml Then the membrane was incubated with Alkaline phosphatase.

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), HRP conjugated)

Predicted band size: 35 kDa

Blocking and Diluting buffer and concentration:5% NFDM/TBST

Exposure time:3 minutes

ICC/IF image of ab32104 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32104, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

ab32104 (2µg/ml) staining PP2A alpha  in human pancreas using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic staining of the islet of Langerhans.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min 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, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling PP2A alpha + beta with purified ab32104 at 1/20 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.

Dot blot was performed using ab32104 at 1/1000 dilution.Goat Anti-Rabbit IgG,(H+L),HRP conjugated (ab97051) was used as secondary antibody at 1/100000 dilution

Lane 1:PP2A alpha + beta  phospho peptide

Lane 2:PP2A alpha + beta non-phospho peptide

Blocking and Diluting buffer and concentration:5% NFDM/TBST

Exposure time:10 seconds

All lanes : Anti-PP2A alpha + beta antibody [E155] (ab32104) at 1/5000 dilution

Lane 1 : untreated A431 cell lysate
Lane 2 : EGF treated A431 cell lysate (the specific EGF concentration and incubation time is confidential information)

Predicted band size: 35 kDa
Observed band size: 35 kDa