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Anti-Phospho-Smad3 (Ser423, Ser425) Polyclonal Antibody

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Catalog No. TMAB-01499

Anti-Phospho-Smad3 (Ser423, Ser425) Polyclonal Antibody is a Rabbit antibody targeting Phospho-Smad3 (Ser423, Ser425). Anti-Phospho-Smad3 (Ser423, Ser425) Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,IF,FCM.

Anti-Phospho-Smad3 (Ser423, Ser425) Polyclonal Antibody

Anti-Phospho-Smad3 (Ser423, Ser425) Polyclonal Antibody

😃Good
Catalog No. TMAB-01499
Anti-Phospho-Smad3 (Ser423, Ser425) Polyclonal Antibody is a Rabbit antibody targeting Phospho-Smad3 (Ser423, Ser425). Anti-Phospho-Smad3 (Ser423, Ser425) Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,IF,FCM.
Pack SizePriceAvailabilityQuantity
50 μL$222 7-10 days
100 μL$374 7-10 days
200 μL$527 7-10 days
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Product Introduction

Bioactivity
Description
Anti-Phospho-Smad3 (Ser423, Ser425) Polyclonal Antibody is a Rabbit antibody targeting Phospho-Smad3 (Ser423, Ser425). Anti-Phospho-Smad3 (Ser423, Ser425) Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,IF,FCM.
Ig Type
IgG
Reactivity
Human,Mouse,Rat,Pig (predicted:Chicken,Dog,Cow,Horse)
Verified Activity
1. Blank control (Black line): HUVEC (Black).
Primary Antibody (green line): Rabbit Anti-Phospho-Smad3 (Ser423 + Ser425) antibody (TMAB-01499)
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 (Red line): Rabbit Anti-Phopho-Smad3 (Ser423+Ser425) antibody at 37°C for 20 min;
Incubation: Anti-Phospho-Smad3 (Ser423/425) Polyclonal Antibody, Unconjugated (TMAB-01499) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody and DAb staining.
4. 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 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01499) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
5. Paraformaldehyde-fixed, paraffin embedded (rat heart); 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 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01499) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
6. 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 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01499) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
7. Blank control: Hela.
Primary Antibody (green line): Rabbit Anti-Phospho-Smad3 (Ser423 + Ser425) antibody (TMAB-01499)
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.
8. Sample:
Lane 1: Cerebrum (Mouse) Lysate at 40 μg
Lane 2: Heart (Mouse) Lysate at 40 μg
Lane 3: Testis (Mouse) Lysate at 40 μg
Lane 4: Skin (Mouse) Lysate at 40 μg
Lane 5: Kidney (Mouse) Lysate at 40 μg
Lane 6: Cerebrum (Rat) Lysate at 40 μg
Lane 7: Testis (Rat) Lysate at 40 μg
Lane 8: Skin (Rat) Lysate at 40 μg
Lane 9: Kidney (Rat) Lysate at 40 μg
Lane 10: Huvec (Human) Cell Lysate at 30 μg
Lane 11: A549 (Human) Cell Lysate at 30 μg
Lane 12: Hela (Human) Cell Lysate at 30 μg
Lane 13: HT1080 (Human) Cell Lysate at 30 μg
Lane 14: A431 (Human) Cell Lysate at 30 μg
Primary: Anti-Phospho-Smad3 (Ser423 + Ser425) (TMAB-01499) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 52 kDa
Observed band size: 54 kDa
9. 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01499) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
10. 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 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01499) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
11. Paraformaldehyde-fixed, paraffin embedded (rat 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-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01499) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
12. 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 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (Phospho-Smad3 (Ser423 + Ser425)) Polyclonal Antibody, Unconjugated (TMAB-01499) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
13. Blank control: HUVEC (Black).
Primary Antibody (green line): Rabbit Anti-Phospho-Jak1 antibody
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 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.
Application
WB,IHC-P,IHC-Fr,IF,FCM
Recommended Dose
WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; IF: 1:100-500; FCM: 1μg/Test
Antibody Type
Polyclonal
Host SpeciesRabbit
Subcellular LocalizationCytoplasm. Nucleus. Note=Cytoplasmic and nuclear in the absence of TGF-beta. On TGF-beta stimulation, migrates to the nucleus when complexed with SMAD4. Through the action of the phosphatase PPM1A, released from the SMAD2/SMAD4 complex, and exported out of the nucleus by interaction with RANBP1. Co-localizes with LEMD3 at the nucleus inner membrane. MAPK-mediated phosphorylation appears to have no effect on nuclear import. PDPK1 prevents its nuclear translocation in response to TGF-beta.
ConstructionHybridoma Polyclonal Antibody
PurificationProtein A purified
AppearanceLiquid
Formulation0.01M TBS (pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol.
Research BackgroundSmad3 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.
Antigen Details
Immunogen
KLH conjugated Synthesised phosphopeptide: human Smad3 around the phosphorylation site of Ser423/425
Antigen Species
Human
Gene ID
Uniprot ID
Biology Area
Response to hypoxia,SMAD family,SMADs,Hypoxia,Cancer,SMADs,Cytoplasmic
Chemical Properties
Stability & Storage
Stability & StorageStore at -20°C or -80°C for 12 months. Avoid repeated freeze-thaw cycles.
TransportShipping with blue ice.

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Please see Inhibitor Handling Instructions for more frequently ask questions. Topics include: how to prepare stock solutions, how to store products, and cautions on cell-based assays & animal experiments, etc
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