Phospho-Smad3 (Ser423) Rabbit Polyclonal Antibody

Blank control (Black line): HUVEC (Black). Primary Antibody (green line): Rabbit Anti-phospho-Smad3 (Ser423) antibody (orb158428), dilution: 3 µg/10^6 cells, Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647, dilution: 1 µg/Test. Protocol, The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at room temperature. The cells were then 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.

Hela cell, 4% Paraformaldehyde-fixed, Triton X-100 at room temperature for 20 min, Blocking buffer (normal goat serum) at 37°C for 20 min, Antibody incubation with (phospho-Smad3 (Ser423)) polyclonal Antibody, Unconjugated (orb158428) 1:100, 90 minutes at 37°C, followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.

Sample: Lane 1: Heart (Mouse) Lysate at 40 ug, Lane 2: Kidney (Mouse) Lysate at 40 ug, Lane 3: Cerebrum (Rat) Lysate at 40 ug, Primary: Anti-phospho-Smad3 (Ser423) (orb158428) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 52 kD, Observed band size: 52 kD.