New ELISA technique for analysis of p53 protein/DNA binding properties.
The p53 tumour suppressor protein is one of the most important topics in cancer research. Its function is associated with the ability to bind DNA in a sequence-specific manner and to operate as a transcription factor. In the present study, we have developed a rapid and reliable method for analysing sequence-specific binding of p53 protein to DNA using a modified enzyme-linked immunosorbent assay (ELISA). In this p53/DNA-ELISA, we use streptavidin-coated microplates to capture biotinylated oligonucleotides containing p53 consensus sequences (p53CON). This newly developed nonradioactive assay allows the detection of p53/DNA complexes using different monoclonal antibodies recognising p53 and has comparable or higher sensitivity to more complicated radioactive methods. Using this method, we can detect binding of endogenous p53 to p53CON and activation of p53 protein for sequence-specific DNA binding. Variations of the basic protocol have also been developed to perform competition experiments and to study p53 binding to natural binding sequences. This modified DNA-ELISA is applicable for screening p53 binding properties from various sources in a short time.
Jagelská
Eva
E
Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, Brno, Czech Republic.
Brázda
Václav
V
Pospisilová
Sarka
S
Vojtesek
Borivoj
B
Palecek
Emil
E
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004278:DNA, Superhelical; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA