Phospho-Smad3 (Ser208) Rabbit Polyclonal Antibody

• Catalog Number: orb6985
• Category: Antibodies
• Description: Phospho-Smad3 (Ser208) Rabbit Polyclonal Antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: FC, IF, IHC-Fr, IHC-P, WB
• Predicted Reactivity: Mouse
• Reactivity: Human, Rat
• Isotype: IgG
• Immunogen: KLH conjugated Synthesised phosphopeptide derived from human Smad3 around the phosphorylation site of Ser208 NL(p-S)PN
• Antibody Type: Primary Antibody
• Concentration: 1mg/ml
• Dilution range: WB=1:500-2000, IHC-P=1:100-500, IHC-F=1:100-500, IF=1:100-500, Flow-Cyt=1μg/Test
• Form/Appearance: Liquid
• Conjugation: Unconjugated
• MW: 47 kDa
• Target: SMAD3
• UniProt ID: P84022
• RRID: AB_10938499
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Buffer/Preservatives: 0.01M TBS (pH7.4) with 1% rAlbumin, 0.02% Proclin300 and 50% Glycerol.
• Alternative names: Smad3(phospho S208); p-Smad3(phospho S208); SMAD3(
• Note: For research use only
• Expiration Date: 12 months from date of receipt.

Blank control: A431. Primary Antibody (green line): Rabbit Anti-Phospho-Smad3 (Ser208) antibody (orb6985), 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. Acquisition of 20000 events was performed.

Paraformaldehyde-fixed, paraffin embedded (Rat brain), Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Smad3 (Ser208)) Polyclonal Antibody, Unconjugated (orb6985) at 1:500 overnight at 4°C, followed by a conjugated secondary for 20 minutes and DAB staining.

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) (orb6985) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 52 kD, Observed band size: 54 kD.