For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome.
Synthetic peptide within Human AKT1 (C terminal) (phospho S473). The exact sequence is proprietary.
Our Abpromise guarantee covers the use of ab66138 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 1 - 2 µg/ml.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|ELISA||Use a concentration of 0.01 - 0.1 µg/ml.|
|WB||Use a concentration of 0.1 - 0.2 µg/ml. Predicted molecular weight: 55 kDa.
Blocking is recommended with BSA. Non-fat dry milk and PBS should be avoided.
|IP||Use a concentration of 2 - 5 µg/ml.|
ab66138 staining AKT1 in Mouse embryonic stem cells by Immunocytochemistry/Immunofluorescence. The cells were 4% PFA fixed and permeabilized in 0.1% Triton prior to blocking in 10% serum for 30 minutes at room temperature. The primary antibody was diluted 1/1000 and incubated with the sample for 12 hours at 4°C. The secondary antibody was an Alexa Fluor® 594-conjugated Goat anti-Rabbit polyclonal (ab150160), diluted 1/1500.
ICC/IF image of ab66138 stained MCF-7 cells. The cells were 100% methanol fixed (5 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 ab66138 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.
ab66138 staining phospho AKT1 in Mouse embryonic tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde, permeabilized with 0.1% Triton and blocked with 10% serum for 1 hour at 22°C. Samples were incubated with primary antibody (1/500 in blocking buffer (1:10)) for 16 hours at 4°C. An Alexa Fluor®488-conjugated Goat anti-rabbit polyclonal (1/1000)ab150077) was used as the secondary antibody.
PC12 cells were incubated at 37°C for 30 minutes with vehicle control (0 µM) and different concentrations of cabergoline (ab120564). Increased expression of AKT1 (phospho S473) (ab66138) in PC12 cells correlates with an increase in cabergoline concentration, as described in literature.
Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab66138 at 1/1000 dilution and ab8227 at 1 µg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.
1µg peptide blotted onto a nitrocellulose membrane, followed by ab66138 at 1:2000 dilution. A: AKT1 (pS473) peptide B: AKT1 non-phosphopeptide C: non-related phosphopeptide
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"