Phospho-Smad5 (S463 + S465) Recombinant Rabbit Monoclonal Antibody [SY09-03] - BSA and Azide free
Catalog# HA750089
Phospho-Smad5 (S463 + S465) Recombinant Rabbit Monoclonal Antibody [SY09-03] - BSA and Azide free
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WB
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IF-Cell
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IHC-P
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Human
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Mouse
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Rat
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ET1605-5
含抗保成分
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unconjugated
Overview
Product Name
Phospho-Smad5 (S463 + S465) Recombinant Rabbit Monoclonal Antibody [SY09-03] - BSA and Azide free
Antibody Type
Recombinant Rabbit monoclonal Antibody
Immunogen
Synthetic phospho-peptide corresponding to residues surrounding Ser463 and 465 of Human Smad5. The immunogen may cross with p-smad1/9.
Product Specificity
Phospho-SMAD/5 (Ser463/465) (SY09-03) Rabbit mAb detects endogenous levels of SMAD5 only when dually phosphorylated at Ser463 and Ser465 and is also predicted to detect SMAD1 when phosphorylated at Ser463 and Ser465. The antibody may be also predicted to detect SMAD9 (SMAD8) when phosphorylated at Ser465 and Ser467. The antibody does not cross-react with other SMAD-related proteins.
Species Reactivity
Human, Mouse, Rat
Validated Applications
WB, IF-Cell, IHC-P
Molecular Weight
Predicted band size: 52 kDa
Positive Control
HeLa treated with 20 ng/mL TGFβ1 for 15 minutes whole cell lysate, Rat brain tissue lysate, mouse brain tissue lysate, HeLa cells treated with 20ng/mL TGFβ1 for 15 minutes, SKOV-3, HepG2, human tonsil tissue, human breast carcinoma tissue, human liver tissue, mouse brain tissue.
Conjugation
unconjugated
Clone Number
SY09-03
Product Features
Form
Liquid
Concentration
1mg/ml
Storage Instructions
Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage Buffer
1*PBS (pH7.4).
Isotype
IgG
Purification Method
Protein A affinity purified.
Application Dilution
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WB
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1:1,000-1:2,000
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IF-Cell
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1:50-1:200
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IHC-P
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1:50-1:200
Target
Function
Smad proteins, the mammalian homologs of the Drosophila Mothers against dpp (Mad) have been implicated as downstream effectors of TGFβ/BMP signaling. Smad1 (also designated Madr1 or JV4-1), Smad5 and mammalian Smad8 (also designated Smad9 or MADH6) are effectors of BMP2 and BMP4 function while Smad2 (also designated Madr2 or JV18-1) and Smad3 are involved in TGFβ and activin-mediated growth modulation. Smad4 (also designated DPC4) has been shown to mediate all of the above activities through interaction with various Smad family members. Smad6 and Smad7 regulate the response to activin/TGFβ signaling by interfering with TGFβ-mediated phosphorylation of other Smad family members.
Background References
1. Nili M et al. Soluble Repulsive Guidance Molecule c/Hemojuvelin Is a Broad Spectrum Bone Morphogenetic Protein (BMP) Antagonist and Inhibits both BMP2- and BMP6-mediated Signaling and Gene Expression. J Biol Chem 285:24783-92 (2010).
2. Wang L et al. Fibromodulin and Biglycan Modulate Periodontium through TGF /BMP Signaling. J Dent Res 93:780-787 (2014).
Sequence Similarity
Belongs to the dwarfin/SMAD family.
Tissue Specificity
Ubiquitous.
Post-translational Modification
Phosphorylated on serine by BMP (bone morphogenetic proteins) type 1 receptor kinase.; Ubiquitin-mediated proteolysis by SMAD-specific E3 ubiquitin ligase SMURF1.
Subcellular Location
Cytoplasm, Nucleus.
Synonyms
DKFZp781C1895 antibody
DKFZp781O1323 antibody
Dwfc antibody
hSmad5 antibody
JV5 1 antibody
JV5-1 antibody
MAD homolog 5 antibody
MAD, mothers against decapentaplegic homolog 5 antibody
MADH 5 antibody
MADH5 antibody
ExpandImages
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☑ Cell treatment (CT)
Western blot analysis of Phospho-Smad5 (S463 + S465) on different lysates with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody (HA750089) at 1/1,000 dilution.
Lane 1: HeLa whole cell lysate
Lane 2: HeLa treated with 20 ng/mL TGFβ1 for 15 minutes whole cell lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 52 kDa
Observed band size: 52 kDa
Exposure time: 2 minutes;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750089) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:100,000 dilution was used for 1 hour at room temperature. -
Western blot analysis of Phospho-Smad5 (S463 + S465) on different lysates with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody (HA750089) at 1/500 dilution.
Lane 1: Rat brain tissue lysate
Lane 2: Mouse brain tissue lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 52 kDa
Observed band size: 52 kDa
Exposure time: 2 minutes;
8% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750089) at 1/500 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature. -
☑ Cell treatment (CT)
Immunocytochemistry analysis of HeLa cells treated with 20ng/mL TGFβ1 for 15 minutes labeling Phospho-Smad5 (S463 + S465) with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody (HA750089) at 1/50 dilution.
Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody (HA750089) at 1/50 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. -
ICC staining of Phospho-Smad5 (S463 + S465) in SKOV-3 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (HA750089, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
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ICC staining of Phospho-Smad5 (S463 + S465) in HepG2 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (HA750089, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
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Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Phospho-Smad5 (S463 + S465) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750089, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-Phospho-Smad5 (S463 + S465) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750089, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-Phospho-Smad5 (S463 + S465) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750089, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-Phospho-Smad5 (S463 + S465) antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750089, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-Smad5 (S463 + S465) antibody (HA750089) at 1/5,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA750089) at 1/5,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Flow cytometric analysis of HeLa+TGFβ1(20ng/mL 15min)(+) cells labeling Phospho-Smad5 (S463 + S465).
Cells were fixed and permeabilized. Then stained with the primary antibody (HA750089, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
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