PRODUCT CODE: ET1605-5

Recombinant Phospho Smad5 (S463/465) Monoclonal Antibody (ET1605-5)

  • Recombinant

Applications

  • WB

  • ICC

  • IHC-P

REACTIVITY

  • Human

  • Mouse

  • Rat

Western blot analysis of Phospho-Smad5(S463/465) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1605-5, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.<br />
 Positive control: <br />
 Lane 1: mouse brain tissue lysate<br />
 Lane 2: rat brain tissue lysate
  • Western blot analysis of Phospho-Smad5(S463/465) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1605-5, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.<br />
 Positive control: <br />
 Lane 1: mouse brain tissue lysate<br />
 Lane 2: rat brain tissue lysate
  • ICC staining of Phospho-Smad5(S463/465) 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 (ET1605-5, 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).
  • ICC staining of Phospho-Smad5(S463/465) 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 (ET1605-5, 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).
  • Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-Phospho-Smad5(S463/465) 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 (ET1605-5, 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.
  • Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using anti-Phospho-Smad5(S463/465) 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 (ET1605-5, 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.
  • Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-Phospho-Smad5(S463/465) 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 (ET1605-5, 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.
  • Immunohistochemical analysis of paraffin-embedded mouse liver tissue using anti-Phospho-Smad5(S463/465) 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 (ET1605-5, 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.
  • Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-Phospho-Smad5(S463/465) 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 (ET1605-5, 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.
Western blot analysis of Phospho-Smad5(S463/465) on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (ET1605-5, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
Positive control:
Lane 1: mouse brain tissue lysate
Lane 2: rat brain tissue lysate

Applications

  • WB

  • ICC

  • IHC-P

REACTIVITY

  • Human

  • Mouse

  • Rat

SPECIFICATIONS

Product Type

Recombinant Rabbit monoclonal primary

Product Name

Recombinant Phospho Smad5 (S463/465) Monoclonal Antibody (ET1605-5)

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding ser463 and 465 of human smad5.

Host

Rabbit

Modification

Phospho

Modification Site

S463/465

Positive Control

Mouse brain tissue lysate, rat brain tissue lysate, SKOV-3, HepG2, human tonsil tissue, human breast carcinoma tissue, human liver tissue, mouse liver tissue, mouse brain tissue.

Conjugation

Unconjugated

Clonality

Monoclonal

Clone Number

SY09-03

PROPERTIES

Form

Liquid

Storage Condition

Store at +4C after thawing. Aliquot store at -20C or -80C. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Concentration

1 ug/ul

PURIFICATION

Protein A affinity purified.

MOLECULAR WEIGHT

58 kDa

Isotype

IgG

APPLICATION DILUTION

  • WB

  • 1:1,000-1:2,000

  • ICC

  • 1:50-1:200

  • IHC-P

  • 1:50-1:200

TARGET

UNIPROT #

PROTEIN NAME

Phospho-Smad5(S463/465)

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; Mothers against decapentaplegic homolog 5 antibody; mothers against decapentaplegic, drosophila, homolog of, 5 antibody; Mothers against DPP homolog 5 antibody; MusMLP antibody; SMA and MAD related protein 5 antibody; SMAD 5 antibody; SMAD family member 5 antibody; SMAD, mothers against DPP homolog 5 antibody; Smad5 antibody; SMAD5_HUMAN antibody

SEQUENCE SIMILARITIES

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.

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.