Anti-Phospho-Smad3 (Ser204) Polyclonal Antibody is a Rabbit antibody targeting Phospho-Smad3 (Ser204). Anti-Phospho-Smad3 (Ser204) Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM.

IgG

Human,Mouse,Rat (predicted:Pig,Cow,Horse,Rabbit,Sheep)

1. Blank control (Black line): HUVEC (Black).
Primary Antibody (green line): Rabbit Anti-phospho-Smad3 (Ser204) antibody (TMAB-01498)
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF647
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min 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.
2. 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 (green line): Rabbit Anti-Phopho-Smad3 (Ser204) antibody (TMAB-01498);
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC;Dilution: 1 μg/test.
3. Paraformaldehyde-fixed, paraffin embedded (Rat uterus); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-Smad3 (Ser204)) Polyclonal Antibody, Unconjugated (TMAB-01498) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
4. Sample:
Lane 1: Heart (Mouse) Lysate at 40 μg
Lane 2: Kidney (Mouse) Lysate at 40 μg
Lane 3: Skin (Rat) Lysate at 40 μg
Primary: Anti-phospho-Smad3 (Ser204) (TMAB-01498) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 52 kDa
Observed band size: 50 kDa
5. Hela cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (phospho-Smad3 (Ser204)) polyclonal Antibody, Unconjugated (TMAB-01498) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nucleus.
6. Blank control: A431. Primary Antibody (green line): Rabbit Anti-phospho-Smad3 (Ser204) antibody (TMAB-01498)
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: Goat anti-rabbit IgG-AF647
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at-20°C. 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.
7. Paraformaldehyde-fixed, paraffin embedded (human skin cancer); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-Smad3 (Ser204)) Polyclonal Antibody, Unconjugated (TMAB-01498) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
8. Paraformaldehyde-fixed, paraffin embedded (mouse kidney); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-Smad3 (Ser204)) Polyclonal Antibody, Unconjugated (TMAB-01498) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
9. Paraformaldehyde-fixed, paraffin embedded (rat kidney); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-Smad3 (Ser204)) Polyclonal Antibody, Unconjugated (TMAB-01498) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
10. Paraformaldehyde-fixed, paraffin embedded (human gastric carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-Smad3 (Ser204)) Polyclonal Antibody, Unconjugated (TMAB-01498) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; ICC/IF: 1:100; IF: 1:100-500; FCM: 1μg/Test

Polyclonal

KLH conjugated Synthesised phosphopeptide: human Smad3 around the phosphorylation site of Ser204

Human

Response to hypoxia,SMAD family,SMADs,Hypoxia,Cancer,SMADs,Cytoplasmic