Anti-Phospho-Smad3 (Ser208) antibody (bs-5617R)

Anti-Phospho-Smad3 (Ser208) antibody

DATASHEET

Host:Rabbit

Target Protein:Phospho-Smad3 (Ser208)

IR:Immunogen Range:NL(p-S)PN

Clonality:Polyclonal

Isotype:IgG

Entrez Gene:4088

Swiss Prot:P84022

Source:KLH conjugated Synthesised phosphopeptide derived from human Smad3 around the phosphorylation site of Ser208:NL(p-S)PN 

Purification:affinity purified by Protein A

Storage:0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol. Shipped at 4℃. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles.

Background:Smad3 is a 50 kDa member of a family of proteins that act as key mediators of TGF beta superfamily signaling in cell proliferation, differentiation and development. The Smad family is divided into three subclasses: receptor regulated Smads, activin/TGF beta receptor regulated (Smad2 and 3) or BMP receptor regulated (Smad 1, 5, and 8); the common partner, (Smad4) that functions via its interaction to the various Smads; and the inhibitory Smads, (Smad6 and 7). Activated Smad3 oligomerizes with Smad4 upon TGF beta stimulation and translocates as a complex into the nucleus, allowing its binding to DNA and transcription factors. Phosphorylation of the two TGF beta dependent serines 423 and 425 in the C terminus of Smad3 is critical for Smad3 transcriptional activity and TGF beta signaling.

Size:100ul

Concentration:1mg/ml

Applications:WB(1:500-2000)
ELISA(1:5000-10000)
IHC-P(1:100-500)
IHC-F(1:100-500)
Flow-Cyt(1μg/Test)
ICC(1:100-500)
IF(1:100-500)

Cross Reactive Species:Human
Mouse
Rat
.

For research use only. Not intended for diagnostic or therapeutic use.

VALIDATION IMAGES

Sample:
Lane 1: Huvec (Human) Cell Lysate at 30 ug
Lane 2: Hela (Human) Cell Lysate at 30 ug
Lane 3: HT1080 (Human) Cell Lysate at 30 ug
Lane 4: A431 (Human) Cell Lysate at 30 ug
Lane 5: A549 (Human) Cell Lysate at 30 ug
Primary: Anti-Phospho-Smad3 (Ser208) (bs-5617R) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 52 kD
Observed band size: 54 kD

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 (Ser208)) Polyclonal Antibody, Unconjugated (bs-5617R) at 1:500 overnight at 4°C, followed by a conjugated secondary (sp-0023) for 20 minutes and DAB staining.

Blank control:A431.
Primary Antibody (green line): Rabbit Anti-Phospho-Smad3 (Ser208) antibody (bs-5617R)
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.