Antibody Type: Rabbit Polyclonal

Application: WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM

Reactivity: Human,Mouse,Rat (predicted:Chicken,Dog,Cow,Horse,Pig)

IgG

Human,Mouse,Rat (predicted:Chicken,Dog,Cow,Horse,Pig)

1. Blank control (black line): Hela.
Primary Antibody (green line): Rabbit Anti-Phospho-Smad2 (Ser465 + Ser467) antibody (TMAB-01496)
Dilution: 1 μg/Test;
Secondary Antibody (white blue line): Goat anti-rabbit IgG-AF488
Dilution: 0.5 μg/Test.
Isotype control (orange line): Normal Rabbit IgG
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.
2. Paraformaldehyde-fixed, paraffin embedded (Mouse testis); 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-Smad2 (Ser465 + Ser467)) Polyclonal Antibody, Unconjugated (TMAB-01496) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
3. 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-Smad2 (Ser465 + Ser467) ) polyclonal Antibody, Unconjugated (TMAB-01496) 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.
4. Sample:
Lane 1: Placenta (Mouse) Lysate at 40 μg
Lane 2: Raw264.7 (Mouse) Cell Lysate at 30 μg
Lane 3: Testis (Rat) Lysate at 40 μg
Lane 4: Hela (Human) Cell Lysate at 30 μg
Lane 5: HT1080 (Human) Cell Lysate at 30 μg
Lane 6: Jurkat (Human) Cell Lysate at 30 μg
Lane 7: HL60 (Human) Cell Lysate at 30 μg
Primary: Anti-Phospho-Smad2 (Ser465 + Ser467) (TMAB-01496) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 60 kDa
Observed band size: 60 kDa
5. Paraformaldehyde-fixed, paraffin embedded (Mouse brain); 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-Smad2 (Ser465 + Ser467)) Polyclonal Antibody, Unconjugated (TMAB-01496) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

WB,IHC-P,IHC-Fr,ICC/IF,IF,FCM

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

Polyclonal

KLH conjugated Synthesised phosphopeptide: human SMAD2 around the phosphorylation site of Ser465/467

Human

4087

TGF,SMADs,SMADs,Cytoplasmic