GST-IVTT pull-down: a fast and versatile in vitro method for validating and mapping protein-protein interactions

Over the past few decades, dozens of in vitro methods have been developed to map, investigate and validate protein-protein interactions. However, most of these approaches are time-consuming and labour-intensive or require specialised equipment or substantial amounts of purified proteins. Here, we describe a fast and versatile research protocol that is suitable for the in vitro analysis of the physical interaction between proteins or for mapping the binding surfaces. The principle of this method is based on the immobilisation of the protein/domain of interest to a carrier followed by its incubation with a labelled putative binding partner, which is generated by a coupled in vitro transcription/translation reaction. Interacting proteins are removed from the carrier, fractionated and visualised by SDS/PAGE autoradiography (or western blotting). This simple and cheap method can be easily carried out in every wet lab.