RESUMEN

Although the distribution of DNA-binding proteins inside the cell nucleus can be analyzed by immunolabeling or by tagging proteins with GFP, we cannot establish whether the protein is bound to DNA or not. Here, we describe a novel approach that allows imaging of the in situ interaction between a GFP-fusion protein and DNA in the cell nucleus, using fluorescence resonance energy transfer (FRET). We used fluorescence lifetime imaging microscopy (FLIM) as a reliable tool to detect protein in contact with DNA. The method was successfully applied to the DNA-binding proteins histone H2B and the glucocorticoid receptor and to the heterochromatin-associated proteins HP1alpha and HP1beta.

Asunto(s)

Núcleo Celular/metabolismo , ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Nucleares/metabolismo , Línea Celular Tumoral , Homólogo de la Proteína Chromobox 5 , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Microscopía Fluorescente/métodos , Protaminas/metabolismo , Unión Proteica/fisiología