Western Blot Analysis of Protein-DNA Complexes Formed during Gel Shift Experiments

Protein-DNA interactions and the identification of DNA-binding proteins have been studied mostly by using the most widely used electrophoretic mobility shift assay (EMSA). This chapter details the Shift-Western blotting method developed and described by Matthias Harbers to analyze further protein-DNA complexes formed during EMSA experiments. Shift-Western blotting is a procedure where the protein and DNA molecules present in a polyacrylamide gel are transferred to stacked membranes. In the first part, a nitrocellulose membrane holds the proteins while double-stranded DNA passes through the nitrocellulose membrane and binds only to a charged membrane positioned underneath. Restrained proteins can then be stained with precise antibodies, while the DNA can be detected using a radioactive label or a nonradioactive detection system. Shift-Western blotting can overcome several supershift experiments’ restrictions and permit the study of intricate protein-DNA complexes containing many protein factors. Furthermore, proteins and/or DNA may be recovered from membranes following the blotting step for additional analysis by other methods.