Detection of Brachyury-regulated Genes by Chromatin Immunoprecipitation. Mesoderm-differentiated BG01V human embryonic stem cells were fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. Brachyury/DNA complexes were immunoprecipitated using 5 µg Goat Anti-Human/Mouse Brachyury Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2085) 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. The VEGF promoter was detected by standard PCR.

Brachyury in Differentiated Human Embryonic Stem Cells. Brachyury was detected in immersion fixed differentiated human embryonic stem cells using 10 µg/mL Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2085) for 3 hours at room temperature. Cells were stained (green) and counter-stained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Brachyury in Embryonic Mouse Notochord. Brachyury was detected in immersion fixed frozen sections of embryonic mouse notochord (E9.5) using 10 µg/mL Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2085) overnight at 4 °C. Tissue was stained with the Northern-Lights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counter-stained with DAPI (blue). View our protocol for Fluorescent IHC Staining of Frozen Tissue Sections.

Detection of Recombinant Human Brachyury by Western Blot. Western blot shows 10 ng of Recombinant Human Brachyury and Recombinant Human TBX6. PVDF Membrane was probed with 0.1 µg/mL of Goat Anti-Human/Mouse Brachyury Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2085) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). A specific band was detected for Brachyury at approximately 26 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 3.

Brachyury in BG01V Human Stem Cells. Brachyury was detected in immersion fixed BG01V human embryonic stem cells differentiated into mesoderm using Goat Anti-Human Brachyury Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2085) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the Northern-Lights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counter-stained with DAPI (blue). Specific staining was localized to nuclei. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.