Sample:Stomach (Mouse) Lysate at 30 ug
Primary: Anti- MADH7/Smad7 (SL0566R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 46 kD
Observed band size: 50 kD
Sample:Spleen(Mouse) Lysate at 30 ug
Primary: Anti- MADH7/Smad7 (SL0566R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 46 kD
Observed band size: 50 kD
Sample:Lymph node (Mouse) Lysate at 30 ug
Primary: Anti- MADH7/Smad7 (SL0566R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 46 kD
Observed band size: 50 kD
Sample:
Lung(Mouse) Lysate at 30 ug
Placenta(Mouse) Lysate at 30 ug
Large intestine(Mouse) Lysate at 30 ug
Primary: Anti- MADH7/Smad7 (SL0566R) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 46 kD
Observed band size: 50 kD
Sample:
Lane 1: SH-SY5Y (Human) Cell Lysate at 30 ug
Lane 2: HepG2 (Human) Cell Lysate at 30 ug
Lane 3: Cerebrum (Mouse) Lysate at 40 ug
Lane 4: Cerebrum (Rat) Lysate at 40 ug
Lane 5: Stomach (Mouse) Lysate at 40 ug
Lane 6: Lung (Mouse) Lysate at 40 ug
Lane 7: Kidney (Mouse) Lysate at 40 ug
Primary: Anti-MADH7/Smad7 (SL0566R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 50 kD
Observed band size: 50 kD
Sample:
Lane 1: Stomach (Mouse) Lysate at 40 ug
Lane 2: Spleen (Mouse) Lysate at 40 ug
Lane 3: Lung (Mouse) Lysate at 40 ug
Lane 4: SH-SY5Y (Human) Cell Lysate at 30 ug
Lane 5: 293T (Human) Cell Lysate at 30 ug
Primary: Anti-MADH7/Smad7 (SL0566R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 50 kD
Observed band size: 50 kD
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 (MADH7) Polyclonal Antibody, Unconjugated (SL0566R) 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 stomach); 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 (MADH7) Polyclonal Antibody, Unconjugated (SL0566R) 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 (MADH7) Polyclonal Antibody, Unconjugated (SL0566R) 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 lung); 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 (Smad7) Polyclonal Antibody, Unconjugated (SL0566R) at 1:600 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.
Tissue/cell: rat kidney 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-Smad7/Smad6 Polyclonal Antibody, Unconjugated(SL0071R) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody(SP-0023) and DAB(SLC0010) staining
U-2OS cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (MADH7/Smad7) polyclonal Antibody, Unconjugated (SL0566R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
U-2OS cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum, SLC0005) at 37°C for 20 min; Antibody incubation with (MADH7/Smad7) polyclonal Antibody, Unconjugated (SL0566R) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue, C02-04002) was used to stain the cell nuclei.
Blank control: SH-SY5Y.
Primary Antibody (green line): Rabbit Anti-MADH7/Smad7 antibody (SL0566R)
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.
|