Sample:
Lane 1: Cerebrum (Mouse) Lysate at 40 ug
Lane 2: Heart (Mouse) Lysate at 40 ug
Lane 3: Testis (Mouse) Lysate at 40 ug
Lane 4: Skin (Mouse) Lysate at 40 ug
Lane 5: Kidney (Mouse) Lysate at 40 ug
Lane 6: Cerebrum (Rat) Lysate at 40 ug
Lane 7: Testis (Rat) Lysate at 40 ug
Lane 8: Skin (Rat) Lysate at 40 ug
Lane 9: Kidney (Rat) Lysate at 40 ug
Lane 10: Huvec (Human) Cell Lysate at 30 ug
Lane 11: A549 (Human) Cell Lysate at 30 ug
Lane 12: Hela (Human) Cell Lysate at 30 ug
Lane 13: HT1080 (Human) Cell Lysate at 30 ug
Lane 14: A431 (Human) Cell Lysate at 30 ug
Primary: Anti-Phospho-Smad3 (Ser423 + Ser425) (SL3425R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 52 kD
Observed band size: 54 kD
Sample:
293T (Human) Cell Lysate at 30 ug
Cerebrum (Mouse) Lysate at 40 ug
Cerebrum (Rat) Lysate at 40 ug
Ovary (Rat) Lysate at 40 ug
HT1080 (Human) Cell Lysate at 30 ug
Jurkat(Human) Cell Lysate at 30 ug
Liver (Rat) Lysate at 40 ug
Primary: Anti- Phospho-Smad3 (Ser423 + Ser425) (SL3425R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 52 kD
Observed band size: 63 kD
Paraformaldehyde-fixed, paraffin embedded (human gastric carcinoma); 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (SL3425R) 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 heart); 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (SL3425R) 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 kidney); 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (SL3425R) 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 skin); 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (SL3425R) 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 (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (SL3425R) 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 testis); 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (SL3425R) 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 kidney); 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (SL3425R) 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 skin); 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (SL3425R) 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (SL3425R) at 1:200 overnight at 4°C, followed by operating according to SP Kit(Rabbit) (sp-0023) instructionsand DAB staining.
Tissue/cell: human liver 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-Smad3(Ser423/425) Polyclonal Antibody, Unconjugated(SL3425R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
Blank control: Hela.
Primary Antibody (green line): Rabbit Anti-Phospho-Smad3 (Ser423 + Ser425) antibody (SL3425R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody : Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
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.
Blank control (Black line): HUVEC (Black).
Primary Antibody (green line): Rabbit Anti-Phospho-Smad3 (Ser423 + Ser425) antibody (SL3425R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
Protocol
The cells were fixed with 4% PFA (10min 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 20,000 events was performed.
Blank control (black line): HL60(black) (The cells were fixed with 2% paraformaldehyde (10 min) , then permeabilized with PBST for 30 min on room temperature)
Primary Antibody (Red line): Rabbit Anti-Phopho-Smad3(Ser423+Ser425) antibody (SL3152R) ;
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (green line): Rabbit IgG .
Secondary Antibody (red line): Goat anti-rabbit IgG-FITC;Dilution: 1μg /test.
Blank control: HUVEC (Black).
Primary Antibody (green line): Rabbit Anti-Phospho-Jak1 antibody (SL3245R)
Dilution: 1μg /10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG .
Secondary Antibody: Goat anti-rabbit IgG-AF647
Dilution: 1μg /test.
Protocol
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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