Human/Mouse/Rat p53 Antibody

Detection of Human p53 by Western Blot. Western blot shows lysates of CEM human T-lymphoblastoid cell line and MCF-7 human breast cancer cell line were mock-treated (-) or exposed (+) to 10 Gy ionizing radiation (IR) and harvested after 1 hour. PVDF membrane was probed with 0.5 µg/mL of Goat Anti-Human/Mouse/Rat p53 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1355), followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). A specific band was detected for p53 at approximately 53 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of p53-regulated Genes by Chromatin Immunoprecipitation. MCF-7 human breast cancer cell line treated with 300 nM camptothecin overnight were fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. p53/DNA complexes were immunoprecipitated using 5 µg Goat Anti-Human/Mouse/Rat p53 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1355) 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 p21 promoter was detected by standard PCR.

p53 in HeLa Human Cell Line. p53 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line using Goat Anti-Human/Mouse/Rat p53 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1355) at 1.7 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 nuclei. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of Human p53 by Simple Western^TM. Simple Western lane view shows lysates of CEM human T-lymphoblastoid cell line and HEK293T human embryonic kidney cell line, loaded at 0.2 mg/mL. A specific band was detected for p53 at approximately 59 kDa (as indicated) using 2.5 µg/mL of Goat Anti-Human/Mouse/Rat p53 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1355) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Western Blot Shows Human p53 Specificity by Using Knockout Cell Line. Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and p53 knockout HeLa cell line (KO). PVDF membrane was probed with 0.25 µg/mL of Goat Anti-Human/Mouse/Rat p53 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1355) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for p53 at approximately 53 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.