PRODUCT CODE: ET1604-12

Smad4 Recombinant Rabbit Monoclonal Antibody [SP05-05] (ET1604-12)

  • Recombinant

Applications

  • WB

  • ICC

  • IF

  • IHC-P

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

Western blot analysis of Smad4 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 (ET1604-12, 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: PC-12 cell lysate<br />
Lane 2: SH-SY5Y cell lysate
  • Western blot analysis of Smad4 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 (ET1604-12, 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: PC-12 cell lysate<br />
Lane 2: SH-SY5Y cell lysate
  • ICC staining of Smad4 in Hela 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 (ET1604-12, 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 Smad4 in MCF-7 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 (ET1604-12, 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 Smad4 in SH-SY5Y 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 (ET1604-12, 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 lung tissue using anti-Smad4 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 (ET1604-12, 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-Smad4 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 (ET1604-12, 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 colon tissue using anti-Smad4 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 (ET1604-12, 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-Smad4 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 (ET1604-12, 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.
  • Flow cytometric analysis of Smad4 was done on SH-SY5Y cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1604-12, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Western blot analysis of Smad4 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 (ET1604-12, 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: PC-12 cell lysate
Lane 2: SH-SY5Y cell lysate

Applications

  • WB

  • ICC

  • IF

  • IHC-P

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

SPECIFICATIONS

Product Type

Recombinant Rabbit monoclonal primary

Product Name

Smad4 Recombinant Rabbit Monoclonal Antibody [SP05-05] (ET1604-12)

Immunogen

Recombinant protein

Host

Rabbit

Positive Control

PC-12 cell lysate, SH-SY5Y cell lysate, Hela, MCF-7, SH-SY5Y, human lung tissue, human breast carcinoma tissue, mouse colon tissue, mouse brain tissue.

Conjugation

Unconjugated

Clonality

Monoclonal

Clone Number

SP05-05

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

60 kDa

Isotype

IgG

APPLICATION DILUTION

  • WB

  • 1:1,000-1:2,000

  • ICC/IF

  • 1:50-1:200

  • IHC-P

  • 1:50-1:200

  • FC

  • 1:50-1:100

TARGET

UNIPROT #

PROTEIN NAME

Smad4

SYNONYMS

(Small) Mothers Against Decapentaplegic antibody; Deleted in Pancreatic Carcinoma 4 antibody; Deleted in Pancreatic Carcinoma antibody; Deleted in pancreatic carcinoma locus 4 antibody; Deletion target in pancreatic carcinoma 4 antibody; DPC 4 antibody; DPC4 antibody; hSMAD4 antibody; JIP antibody; MAD homolog 4 antibody; MAD mothers against decapentaplegic Drosophila homolog 4 antibody; MAD mothers against decapentaplegic homolog 4 antibody; MADH 4 antibody; MADH4 antibody; Med antibody; Medea antibody; Mothers against decapentaplegic homolog 4 antibody; Mothers against decapentaplegic, Drosophila, homolog of, 4 antibody; Mothers against DPP homolog 4 antibody; MYHRS antibody; OTTHUMP00000163548 antibody; SMA- and MAD-related protein 4 antibody; SMAD 4 antibody; SMAD family member 4 antibody; SMAD mothers against DPP homolog 4 antibody; SMAD4 antibody; SMAD4_HUMAN antibody

SEQUENCE SIMILARITIES

Belongs to the dwarfin/SMAD family.

POST-TRANSLATIONAL MODIFICATION

Phosphorylated by PDPK1.; Monoubiquitinated on Lys-519 by E3 ubiquitin-protein ligase TRIM33. Monoubiquitination hampers its ability to form a stable complex with activated SMAD2/3 resulting in inhibition of TGF-beta/BMP signaling cascade. Deubiquitination by USP9X restores its competence to mediate TGF-beta signaling.

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.