Detection of Phosphorylation-Dependent Interactions by Far-Western Gel Overlay

Abstract

The far Western gel overlay assay is a highly sensitive tool for the detection of direct protein-protein interactions. The ability to distinguish direct associations between two proteins makes this technique ideal not only for identifying potential binding partners of a protein, but also for characterizing interaction domains and binding sites. The assay relies on the ability of the protein of interest to interact with target proteins that have been immobilized on nitrocelulose filters. Potential interactions can be monitored under various types of conditions by manipulation of the protein that is used as the probe. Such manipulations may include mutagenesis, or protein modifications such as phosphorylation. Detection of phosphorylation-dependent interactions is particularly relevant in light of the fact that many signaling proteins are modified by phosphorylation, which in turn affects activity, localization, and most importantly, the ability to form protein complexes (see refs. 1–4). We describe here the standard protocol for the far-Western gel overlay assay, with modifications that enable the detection of phosphorylation-dependent protein interactions. This technique has been used successfully by our laboratory, as well as others to identify potential phosphorylation-dependent interactions between a number of signaling proteins (5–7).