Question

In-vivo method to identify protein- dna interaction

Answer 1

Many events in the viral life cycle involve protein binding to defined sequences on the viral chromosome. Chromatin immunoprecipitation allows the detection of the in vivo interaction of specific proteins with specific genomic regions. In this technique, living cells are treated with formaldehyde to crosslink neighboring protein-protein and protein-DNA molecules. The crosslink with formaldehyde is reversible and covers a short distance (2 A); the components that are crosslinked are therefore in close proximity. Nuclear fractions are isolated, and the genomic DNA is sheared to reduce the average DNA fragment size to around 500 bp. These nuclear lysates are used in immunoprecipitations with an antibody against the protein of interest. The DNA bound to the studied protein is enriched after the immunoprecipitation. After reversal of the crosslinking, the resulting DNA and proteins can be independently studied. The electrophoretic mobility shift assay provides a rapid method to study DNA-binding protein interactions in vitro.