All lanes : Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505) at 1/10000 dilution

Lane 1 : 293T cell lysate transfected with GFP tagged AKT1
Lanes 2 & 4 : 293T cell lysate transfected with empty vector
Lane 3 : 293T cell lysate transfected with GFP tagged AKT3

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 56 kDa
Observed band size: 82 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

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

ab32505 staining in SK-N-SH cells treated with alsterpaullone (ab141070), by ICC/IF. Decrease of AKT1 + AKT2 + AKT3 expression correlates with increased concentration of alsterpaullone, as described in literature.
The cells were incubated at 37°C for 6h in media containing different concentrations of ab141070 (alsterpaullone) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab32505 (1/200 dilution was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

Immunohistochemical analysis of paraffin-embedded prostate carcinoma using ab32505 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Purified ab32505 at 1/50 dilution (2µg) immunoprecipitating AKT3+AKT2+AKT1 in MCF7 whole cell lysate.
Lane 1 (input): MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab32505 + MCF7 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32505 in MCF7 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 59 kDa

All lanes : Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505) at 1/2000 dilution

Lane 1 : 293T cell lysate transfected with GFP tagged AKT2
Lane 2 : 293T cell lysate transfected with empty vector

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 56 kDa
Observed band size: 82 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

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

Anti-AKT3 + AKT2 + AKT1 antibody [Y89] (ab32505) at 1/10000 dilution + MCF-7 cell lysate

Predicted band size: 56 kDa
Observed band size: 59 kDa why is the actual band size different from the predicted?

Overlay histogram showing HeLa cells stained with ab32505 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32505, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.