Human/Mouse/Rat RelA/NF kappa B p65 Antibody

Detection of Human RelA/NF kappa B p65 by Western Blot. Western blot shows lysates of K562 human chronic myelogenous leukemia cell line, Daudi human Burkitt's lymphoma cell line, and LNCaP human prostate cancer cell line. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human/Mouse/Rat RelA/NF kappa B p65 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5078) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for RelA/NF kappa B p65 at approximately 70 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human and Mouse RelA/NF kappa B p65 by Western Blot. Western blot shows lysates of K562 human chronic myelogenous leukemia cell line, HeLa human cervical epithelial carcinoma cell line, and Neuro-2A mouse neuroblastoma cell line. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human/Mouse/Rat RelA/NF kappa B p65 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5078) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for RelA/NF kappa B p65 at approximately 70 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Mouse and Rat RelA/NF kappa B p65 by Western Blot. Western blot shows lysates of NIH-3T3 mouse embryonic fibroblast cell line and C6 rat glioma cell line. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human/Mouse RelA/NF kappa B p65 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5078) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for RelA/NF kappa B p65 at approximately 65 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of RelA/NF kappa B p65-regulated Genes by Chromatin Immuno-precipitation. Jurkat human acute T cell leukemia cell line treated with 50 ng/mL PMA and 200 ng/mL calcium ionomycin for overnight was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. RelA/NF kappa B p65/DNA complexes were immunoprecipitated using 5 µg Sheep Anti-Human/Mouse/Rat RelA/NF kappa B p65 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5078) or control antibody (Catalog # 5-001-A) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Sheep IgG Secondary Antibody (Catalog # [[catalogNumber:BAF016)]]. Immunocomplexes were captured using 50 µL of MagCellect Streptavidin Ferrofluid (Catalog # [[catalogNumber:MAG999]]) and DNA was purified using chelating resin solution. Thep21promoter was detected by standard PCR.

RelA/NF kappa B p65 in HeLa Human Cell Line. RelA/NF kappa B p65 was detected in immersion fixed HeLa human cervical epithelial carcinoma cells untreated (upper panel) or treated (lower panel) with 20 ng/mL Recombinant Human TNF-alpha (Catalog # 210-TA) for 10 minutes using Sheep Anti-Human/Mouse/Rat RelA/NF kappa B p65 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5078) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm in untreated cells and nuclei in treated cells. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

RelA/NF kappa B p65 in Human Squamous Cell Carcinoma. RelA/NF kappa B p65 was detected in immersion fixed paraffin-embedded sections of human squamous cell carcinoma using Sheep Anti-Human/Mouse/Rat RelA/NF kappa B p65 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5078) at 3 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Sheep HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS019) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in cancer cells. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Human, Mouse, and Rat RelA/NF kappa B p65 by Simple Western^TM. Simple Western lane view shows lysates of HeLa human cervical epithelial carcinoma cell line, Neuro-2A mouse neuroblastoma cell line, and C6 rat glioma cell line, loaded at 0.5 mg/mL. A specific band was detected for RelA/NF kappa B p65 at approximately 65 kDa (as indicated) using 20 µg/mL of Sheep Anti-Human/Mouse/Rat RelA/NF kappa B p65 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5078) followed by 1:50 dilution of HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western Blot Shows Human RelA/NF kappa B p65 Specificity by Using Knockout Cell Line. Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and RelA/NF kappa B p65 knockout HeLa cell line (KO). PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human/Mouse/Rat RelA/NF kappa B p65 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5078) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for RelA/NF kappa B p65 at approximately 65 kDa (as indicated) in the parental HeLa cell line, but is not detectable in knockout HeLa cell line. GAPDH (Catalog # AF5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.