Biochemical and Immunological Detection of Physical Interactions Between Penta-EF-Hand Protein ALG-2 and Its Binding Partners

Many nonenzymatic cellular proteins exert their functions by interacting with other proteins or -macromolecules. Analysis of the physical interactions of proteins is an important step to understand their functions, and the information obtained is helpful for predicting the roles of the proteins in cells. Here we describe three biochemical and immunological methods for the detection of interactions between ALG-2 (a penta-EF-hand Ca(2+)-binding protein, also known as PDCD6) and its target proteins: (1) glutathione-S-transferase (GST) pulldown assay, (2) co-immunoprecipitation assay, and (3) Far Western blot analysis using biotinylated ALG-2. Dependency of Ca(2+) for interaction is examined by inclusion of CaCl(2) or EGTA in buffers used for binding assays.