Sample:
Hela(Human) Cell Lysate at 30 ug
293T(Human) Cell Lysate at 30 ug
Primary: Anti-PERK (bs-2469R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 122 kD
Observed band size: 122 kD

Sample:
Lane 1: Cerebrum (Mouse) Lysate at 40 ug
Lane 2: Cerebrum (Rat) Lysate at 40 ug
Primary: Anti-PERK (bs-2469R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 150 kD
Observed band size: 145 kD

Tissue/cell: mouse stomach 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,C-0005) at 37℃ for 20 min;
Incubation: Anti-PERK Polyclonal Antibody, Unconjugated(bs-2469R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(C-0010) staining

Paraformaldehyde-fixed, paraffin embedded (mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (PERK) Polyclonal Antibody, Unconjugated (bs-2469R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30min; Antibody incubation with (PERK) Polyclonal Antibody, Unconjugated (bs-2469R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.

Blank control: U-87MG(blue).
Primary Antibody:Rabbit Anti-PERK antibody(bs-2469R), Dilution: 1μg in 100 μL 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.
Protocol
The cells were fixed with 2% paraformaldehyde (10 min) , then permeabilized with 90% ice-cold methanol for 30 min on ice. Primary antibody (bs-2469R,1μg /1x10^6 cells) were incubated for 30 min on the ice, followed by 1 X PBS containing 0.5% BSA + 1 0% goat serum (15 min) to block non-specific protein-protein interactions. Then the Goat Anti-rabbit IgG/PE antibody was added into the blocking buffer mentioned above to react with the primary antibody at 1/200 dilution for 30 min on ice. Acquisition of 20,000 events was performed.

Blank control（black line）:K562.
Primary Antibody (green line): Rabbit Anti-PERK antibody (bs-2469R)
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.