Chromatin Immunoprecipitation (ChIP)
|Detection of STAT3-regulated Genes by Chromatin Immunoprecipitation. Jurkat human acute T cell leukemia cell line treated with 1000 U/mL Recombinant Human IFN‑ alpha Universal Type I (Catalog # 11200-1) for 1 hour was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. STAT3/DNA complexes were immunoprecipitated using 5 μg Goat Anti-Human/Mouse/Rat STAT3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1799) 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 c-myc promoter was detected by standard PCR.|
|Immunoprecipitation of Human STAT3. STAT3 was immunoprecipitated from 500 μg of Daudi human Burkitt's lymphoma cell line cytoplasmic and nuclear lystates following incubation with 2 µg Goat Anti-Human/Mouse/Rat STAT3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1799) for overnight at 4 ºC. STAT3-antibody complexes were absorbed using Protein G agarose (Invitrogen). Immunoprecipitated STAT3 was detected by Western blot using 0.1 µg/mL Human STAT3 Monoclonal Antibody (Catalog # MAB1799). View our recommended buffer recipes for immunoprecipitation.|
|STAT3 in D3 Mouse Cell Line. STAT3 was detected in immersion fixed D3 mouse embryonic stem cell line using Goat Anti-Human/Mouse/Rat STAT3 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1799) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001). Tubulin was also detected using Tubulin Antibody (Catalog # NB600-506; Novus Biologicals) and stained using the NorthernLights™ 493-conjugated Anti-Rat IgG Secondary Antibody (green; Catalog # NL015). Cells were counterstained with DAPI (blue). Specific staining of STAT3 was localized to nuclei. View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips.|
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