Homogeneous time-resolved fluorescence assay for identifying p53 interactions with its protein partners, directly in a cellular extract.
The p53 protein is a tumor suppressor that protects the organism against malignant consequences of DNA damage. Interaction of p53 with numerous cellular or viral proteins regulates its functional activity either positively or negatively. An approach leading to identification of such protein interactions directly in a cell extract could be of help in the development of screening assays to search for drugs acting on p53 in its cellular environment, either by disrupting its association with inhibitory proteins or by increasing its affinity for activating proteins. We show that the homogeneous time-resolved fluorescence (HTRF) assay based on the time-resolved amplified cryptate emission (TRACE) technology allows identification of such an interaction by simply adding a mixture of two labeled monoclonal antibodies, directly in a cellular extract. We validate this assay by studying p53/SV40-LTAg interactions. The antibodies directed against genuine p53 and SV40-LTAg epitopes were labeled with europium cryptate (donor) and XL665, a crosslinked allophycocyanin (acceptor), respectively. We demonstrated that a nonradiative energy transfer occurs between labeled antibodies only when p53 interacts with SV40-LTag, which opens up the possibility of extending this approach to other p53 partners to search for drugs that restore p53 tumor-suppressor activity.
Leblanc
Virginie
V
CEA, CNRS, Laboratoire de Cancérogenèse Moléculaire, UMR217, DRR, DSV, CEA, Fontenay-aux-Roses, France.
Delaunay
Valérie
V
Claude Lelong
Jean
J
Gas
Fabienne
F
Mathis
Gérard
G
Grassi
Jacques
J
May
Evelyne
E
D000818:Animals; D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D019556:COS Cells; D004797:Enzyme-Linked Immunosorbent Assay; D015200:Fluoroimmunoassay / Q000379:methods*; D000882:Haplorhini; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53