Sample:Brain(Mouse) lysate at 30ug;
Primary: Anti-phospho-PKR (Thr446) (SL3335R) at 1:300 dilution;
Secondary: HRP conjugated Goat Anti-Rabbit IgG(SL0295G-HRP) at 1: 5000 dilution;
Predicted band size : 62kD
Observed band size : 62kD
Sample:
Lane 1: Human K562 cell lysates
Lane 2: Human A431 cell lysates
Primary: Anti-Phospho-PKR (Thr446) (SL3335R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 62 kDa
Observed band size: 60 kDa
Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer ( 0.01M, pH 6.0 ), Boiling bathing for 15min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30min; Blocking buffer (normal goat serum,SLC0005) at 37℃ for 20 min;
Incubation: Anti-Phospho-PKR (Thr446) Polyclonal Antibody, Unconjugated(SL3335R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Blank control(black line):Hela.
Primary Antibody (green line): Rabbit Anti-Phospho-PKR (Thr446) antibody (SL3335R)
Dilution:2ug/Test;
Secondary Antibody(white blue line): Goat anti-rabbit IgG-AF488
Dilution: 0.5ug/Test.
Isotype control(orange line): Normal Rabbit IgG
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.
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