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Blank control(blue): Hela(fixed with 2% paraformaldehyde (10 min), then permeabilized with 90% ice-cold methanol for 30 min on ice). Primary Antibody:Rabbit Anti- phospho-ASK1 (Thr845) antibody(bs-3031R), Dilution: 0.2ug in 100 uL 1X PBS containing 0.5% BSA; Isotype Control Antibody: Rabbit IgG(orange),used under the same conditions ); Secondary Antibody: Goat anti-rabbit IgG-PE(white blue), Dilution: 1:200 in 1 X PBS containing 0.5% BSA.
Sample: Lane1: Brain(Rat) Lysate at 30 ug Lane2: Heart(Rat) Lysate at 30 ug Primary: Anti-phospho-ASK1(Thr845) at 1:200 dilution; Secondary: HRP conjugated Goat-Anti-Rabbit IgG at 1: 3000 dilution; Predicted band size : 155kD Observed band size : 155kD
Sample: Lane 8: Cerebrum (Rat) Lysate at 40 ug Lane 9: Stomach (Rat) Lysate at 40 ug Primary: Anti-phospho-ASK1 (Thr845) at 1/1000 dilution Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution Predicted band size: 154 kD Observed band size: 154 kD
Blank control:Mouse spleen. Primary Antibody (green line): Rabbit Anti-phospho-RelB (Ser551) antibody Dilution: 2ug/10^6 cells; Isotype Control Antibody (orange line): Rabbit IgG. Secondary Antibody : Goat anti-rabbit IgG-AF488 Dilution: 1ug/T. Protocol The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 90% ice-cold methanol for 20 min at -20℃. 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 20,000 events was performed.
