In recent years, various methods have been developed to study protein-protein interactions (PPI). PPI plays an important role in the cell signaling cascade; for example, dephosphorylation of glycogen synthase by protein phosphatase-1 results in glycogen synthesis. To know whether a specific protein binds to its partner, for example, whether TFIIH interacts with TFIIE or TFIIF to complete the pre-initiation complex in transcription, several methods such as co-immunoprecipitation (co-IP), glutathione-S -transferase (GST), pull down assay, yeast-two-hybrid (Y2H) assay, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) spectroscopy and so on can be used to validate PPIs. However, performing an experiment using one method is not sufficient to validate the PPI between two or more proteins. Factors such as overexpression of proteins and manipulation of agents used in the experiment could result in skewed data. Therefore, the results should be unbiased by incorporating different methods into the experiment to validate the PPI. In this essay we will describe the different methods and discuss the factors that cause the different methods to give rise to different results. Co-IP is the most commonly used method to test protein-protein interactions (Berggård et al., 2007). Antibodies specific to the bait complexes are used to capture the bait complexes in a cell lysate shown in Fig. 1. The antibody is immobilized on protein A/G, which is covalently bound to the agarose beads. Since the antibody is specific only to the bait complex, the antibody will not bind to other proteins present in the cell lysate and, therefore, these proteins will be removed. The complete antibody bait...... middle of paper ......Inflammatory arthritis in mice. Science. 332 (6028), pp. 478-484. Wissmueller S., Font J., Liew CW, Cram E., Schroeder T., Turner J., Crossley M., Mackay JP, & Matthews JM (2011). Protein-protein interactions: Analysis of a false-positive GST pulldown result. Proteins. 79 (8), pp. 2365-2371. Yu H. (1999). Extending the size limit of protein nuclear magnetic resonance. Proceedings of the National Academy of Sciences. 96 (2), pp. 332-334. Zhang X., Tang H., Ye C., and Liu M. (2006). Structure-based drug design: NMR-based approach for ligand-protein interactions. Drug discovery today: technologies. 3 (3), pp. 241-245. Zhou YL., Liao JM., Du F. and Liang Yi. (2005). Thermodynamics of the interaction of xanthine oxidase with superoxide dismutase studied by isothermal titration calorimetry and fluorescence spectroscopy. Termochimica Acta. 426 (1-2), pp. 173-178.