Key Product Details

Validated by

Knockout/Knockdown, Biological Validation

Species Reactivity

Validated:

Human

Cited:

Human

Applications

Validated:

Western Blot, Immunocytochemistry, Chromatin Immunoprecipitation (ChIP)

Cited:

Western Blot, Immunocytochemistry, Simple Western, Immunoprecipitation, Chromatin Immunoprecipitation (ChIP)

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all Smad4 Primary Antibodies »

Product Specifications

Immunogen

E. coli-derived recombinant human Smad4
Pro139-Asp332
Accession # Q13485

Specificity

Detects human Smad4 in direct ELISAs and Western blots. In direct ELISAs and Western blots, approximately 15% cross-reactivity with recombinant human (rh) Smad1 and less than 5% cross-reactivity with rhSmad9, rhSmad5, and rhSmad7 is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Human Smad4 Antibody

Detection of Human Smad4 antibody by Western Blot.

Detection of Human Smad4 by Western Blot.

Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line, Jurkat human acute T cell leukemia cell line, and K562 human chronic myelogenous leukemia cell line. PVDF membrane was probed with 0.25 µg/mL of Goat Anti-Human Smad4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2097) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). A specific band was detected for Smad4 at approximately 60 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Smad4-regulated Genes antibody by Chromatin Immunoprecipitation.

Detection of Smad4-regulated Genes by Chromatin Immunoprecipitation.

Jurkat human acute T cell leukemia cell line treated with 10 ng/mL Recombinant Human IL-12 (Catalog # 219-IL) overnight was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. Smad4/DNA complexes were immunoprecipitated using 5 µg Goat Anti-Human Smad4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2097) or control antibody (Catalog # AB-108-C) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Goat IgG Secondary Antibody (Catalog # BAF109). Immunocomplexes were captured using 50 µL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. Thep21promoter was detected by standard PCR.

Smad4 antibody in Human PBMCs by Immunocytochemistry (ICC).

Smad4 in Human PBMCs.

Smad4 was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) using Goat Anti-Human Smad4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2097) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Non-adherent Cells.

Detection of Human Smad4 by Western Blot

Detection of Human Smad4 by Western Blot

siRNA knockdown of Smad1, -2 and 4 does not impact RVFV replication.HSAECs transfected with media alone (Mock) or siRNAs (75nM) directed against a nontargeting control (NTC), Smad1, -2, or -4 were infected with MP12 virus (MOI 0.1). Both extracellular media supernatants (A) and protein lysates (B,C) were harvested at 9 and 24hpi. A) Infectious viral titers were determined by plaque assay. Data plotted as a bar graph of the mean with standard deviation of four replicates. Protein lysates from single (B) and double knockdowns (C) were analyzed by western blot for actin, total Smad1, -2, and -4 expression. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29408900), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human Smad4 by Western Blot

Detection of Human Smad4 by Western Blot

siRNA knockdown of Smad1, -2 and 4 does not impact RVFV replication.HSAECs transfected with media alone (Mock) or siRNAs (75nM) directed against a nontargeting control (NTC), Smad1, -2, or -4 were infected with MP12 virus (MOI 0.1). Both extracellular media supernatants (A) and protein lysates (B,C) were harvested at 9 and 24hpi. A) Infectious viral titers were determined by plaque assay. Data plotted as a bar graph of the mean with standard deviation of four replicates. Protein lysates from single (B) and double knockdowns (C) were analyzed by western blot for actin, total Smad1, -2, and -4 expression. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29408900), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human Smad4 Antibody

Application
Recommended Usage

Chromatin Immunoprecipitation (ChIP)

5 µg/5 x 106 cells
Sample: Recombinant Human IL-12 (Catalog # 219-IL) treated Jurkat human acute T cell leukemia cell line chromatin, p21 promoter detected by standard PCR

Immunocytochemistry

5-15 µg/mL
Sample: Immersion fixed human peripheral blood mononuclear cells (PBMCs)

Western Blot

0.25 µg/mL
Sample: HeLa human cervical epithelial carcinoma cell line, Jurkat human acute T cell leukemia cell line, and K562 human chronic myelogenous leukemia cell line

Reviewed Applications

Read 1 review rated 5 using AF2097 in the following applications:

Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Smad4

Smads are a family of intracellular proteins that transmit transforming growth factor beta (TGF-beta ) superfamily signals from the cell surface to the nucleus. Upon signal-induced phosphorylation, Smad subunits associate with the common-mediator subunit, Smad4. This heteromeric complex then translocates into the nucleus to exert transcriptional comodulator activity.

Long Name

Mothers Against DPP Homolog 4

Alternate Names

DPC4, MADH4

Entrez Gene IDs

4089 (Human)

Gene Symbol

SMAD4

UniProt

Q13485

Additional Smad4 Products

Product Documents for Human Smad4 Antibody

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Human Smad4 Antibody

For research use only

Citations for Human Smad4 Antibody

Customer Reviews for Human Smad4 Antibody (1)

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  • Human Smad4 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: Breast cancer cells
    Species: Human
    Verified Customer | Posted 03/16/2017
    Western Blot of breast cancer cell lysates. The PVDF membrane was probed with 1:1,000 Goat anti-Human Smad4 antibody (#AF2097) followed by 1:5,000 anti-Goat secondary antibody.
    Human Smad4 Antibody AF2097

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