Use of gel retardation to analyze protein-DNA interactions upstream of CYPIA1 gene

This chapter discusses the use of gel retardation to analyze protein-DNA interactions upstream of CYPIA1 gene. The gel retardation or gel mobility shift technique has become a standard tool for analyzing the interaction in vitro between DNA-binding proteins and their cognate genomic recognition sequences. The method relies on the fact that, during electrophoresis under nondenaturing conditions, protein-DNA complexes have a reduced mobility compared with protein-free DNA. Several groups have used gel retardation to analyze tetrachlorodibenzo-p-dioxin (TCDD)-inducible protein–DNA interactions at regulatory domains upstream of the CYPIA1 gene. The conditions used by each group vary somewhat, revealing some flexibility in the assay conditions. Gel retardation is also used to analyze cytosolic proteins from both mouse hepatoma cells and rodent liver. The cytosolic preparations are stored under the same conditions as the nuclear extracts, and exhibit similar stabilities.