Blank control: A549. Primary Antibody (green line): Rabbit Anti-phospho-AKT1 (Tyr474) antibody (orb155627), Dilution: 2 µg/10 6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody: Goat anti-rabbit IgG-FITC, Dilution: 1 µg/Test. Protocol, The cells were incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed.
Sample: Hela (Human) Cell Lysate at 30 ug, Primary: Anti-phospho-AKT1 (Tyr474) (orb155627) at 1/300 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 56 kD, Observed band size: 56 kD.
Sample: Lung (Mouse) Lysate at 40 ug, Primary: Anti-phospho-AKT1 (Tyr474) (orb155627) at 1/300 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 56 kD, Observed band size: 56 kD.
Tissue/Cell: A549 cell, 4% Paraformaldehyde-fixed, Triton X-100 at room temperature for 20 min, Blocking buffer (normal goat serum) at 37C for 20 min, Antibody incubation with (phospho-AKT1 (Tyr474)) polyclonal Antibody, Unconjugated (orb155627) 1:100, 90 minutes at 37C, followed by a FITC conjugated Goat Anti-Rabbit IgG antibody at 37C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.
Tissue/Cell: human myometrium tissue, 4% Paraformaldehyde-fixed and paraffin-embedded, Antigen retrieval: citrate buffer (0.01M, pH 6.0), Boiling bathing for 15 min, Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min, Blocking buffer (normal goat serum) at 37C for 20 min, Incubation: Anti-phospho-AKT1 (Tyr474) Polyclonal Antibody, Unconjugated (orb155627) 1:200, overnight at 4C, followed by conjugation to the secondary antibody and DAB staining.
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