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 Ca2+-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 Ca2+ for interaction is examined by inclusion of CaCl2 or EGTA in buffers used for binding assays.
Key wordsProtein–protein interaction Calcium-binding protein GST-pulldown Co-immuno-precipitation Far Western Biotinylation, EF-hand
This work was supported by a Grant-in-Aid for Scientific Research (B) (to MM). We thank members of the Laboratory of Molecular and Cellular Regulation for valuable suggestions.
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