A monoclonal antibody against DNA binding helix of p53 protein

Abstract

Three monoclonal antibodies (Mabs) were generated against p53 DNA-binding core domain. When tested by immunoprecipitation, Western blot and immunofluorescence techniques, Mab 9E4, as well as 7D3 and 6B10 reacted with both wild-type and various mutant p53 proteins. The epitopes recognized by Mabs 7D3, 9E4 and 6B10 were located respectively within the amino acid residues 211-220, 281-290 and 291-300 of human p53 protein. The epitope recognized by 9E4 Mab coincides with helix 2, also called p53 DNA binding helix, which allows the direct contact of the protein with its target DNA sequences. This antibody may be useful to study transcription-dependent and transcription-independent activities of wild-type and mutant p53 proteins

NAPO as a novel marker for apoptosis

“…FITC-conjugated goat anti–mouse antibody (Dako) was used as the secondary antibody and diluted as recommended by the supplier. The immunofluorescence staining of Huh7 cells for p53 protein was tested using 6B10 monoclonal antibody ( Yolcu et al, 2001 ). Nuclear DNA was visualized by incubation with 3 μg/ml Hoechst 33258 (Sigma-Aldrich) for 5 min in the dark.…”

Reprogramming of replicative senescence in hepatocellular carcinoma-derived cells

“…Antibodies against cyclin D1, CDK4, CDK2, p21 Cip1 , pRb (all from Santa Cruz Biotechnology), cyclin E (Transduction), cyclin A (Abcam), p16 INK4a (Abcam), p53 (clone 6B10; ref. 35), and calnexin (Sigma) were used for immunoblotting as described (33).…”

p53 mutation as a source of aberrant β-catenin accumulation in cancer cells

“…USA) and GSK-3b (clone 7 from Transduction Labs., USA) were obtained commercially. The anti-p53 monoclonal antibody 6B10 was described previously (Yolcu et al, 2001). In order to generate a linear response in Western blotting assays, we used appropriate amounts of proteins from cell lysates for SDS -PAGE and an ECL kit (Amersham Pharmacia Biotech, UK) for detection of antigen-antibody complexes.…”