Sample:
Heart (Mouse) Lysate at 30 ug
SP2/0 Cell Lysate at 30 ug
Primary: Anti-phospho-ERK1(Thr202/Tyr204)+ERK2(Thr183/Tyr185) (SL1646R) at 1:200 dilution;
Secondary: HRP conjugated Goat Anti-Rabbit IgG(SL0295G-HRP) at 1: 3000 dilution;
Predicted band size : 42kD
Observed band size : 42kD
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 (phospho-ERK1 (Thr202Tyr204) + ERK2 (Thr183Tyr185)) Polyclonal Antibody, Unconjugated (SL1646R) 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 pancreas); 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 (phospho-ERK1 (Thr202Tyr204) + ERK2 (Thr183Tyr185)) Polyclonal Antibody, Unconjugated (SL1646R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse pancreas); 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 (phospho-ERK1 (Thr202Tyr204) + ERK2 (Thr183Tyr185)) Polyclonal Antibody, Unconjugated (SL1646R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB 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 (phospho-ERK1 (Thr202Tyr204) + ERK2 (Thr183Tyr185)) Polyclonal Antibody, Unconjugated (SL1646R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Paraformaldehyde-fixed, paraffin embedded (mouse brain tissue); 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 (P-ERK 1+ ERK 2) Polyclonal Antibody, Unconjugated (SL1646R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Paraformaldehyde-fixed, paraffin embedded (rat lung tissue); 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 (P-ERK 1+ ERK 2) Polyclonal Antibody, Unconjugated (SL1646R) at 1:400 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Tissue/cell: human lung carcinoma; 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-ERK1(Thr202/Tyr204)+ERK2(Thr183/Tyr185) Polyclonal Antibody, Unconjugated(SL1646R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
From 《Cancer Medicine》(2016.6): PublitionDirect effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes , IF:2.5
Author: Noriyoshi Iriyama, Yoshihiro Hatta & Masami Takei
Division of Hematology and Rheumatology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan
Blank control: MCF7.
Primary Antibody (green line): Rabbit Anti-Phospho-ERK1 (Thr202/Tyr204) + ERK2 (Thr183/Tyr185) antibody (SL1646R)
Dilution: 2μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-FITC
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min at room temperature)and then permeabilized with 0.1% PBST 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 20,000 events was performed.
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