As biotechnology advances and our understanding of protein-protein interactions expands, scientists are utilizing different techniques to characterize and study these interactions. In this post, we will compare traditional and advanced techniques for characterizing protein-protein interactions in a factual and unbiased manner.
Traditional Techniques
Traditional techniques for studying protein-protein interactions include co-immunoprecipitation (Co-IP) and pull-down assays. Co-IP involves the use of antibodies to immunoprecipitate one protein from a mixture, along with any interacting proteins. The pulled-down proteins are then analyzed by western blotting or mass spectrometry to identify the interacting proteins. Similarly, pull-down assays involve immobilizing one protein in a column and then incubating it with a cell lysate to allow interacting proteins to bind. The column is then washed to remove non-specifically bound proteins, and the bound proteins are analyzed to identify the interacting proteins.
While these traditional techniques have been widely used, they have some drawbacks. Both techniques require the prior knowledge of one of the interacting partners, making it challenging to identify novel interactions. Furthermore, these techniques have a low throughput, requiring a lot of time and effort to analyze a small number of interactions.
Advanced Techniques
In recent years, several advanced techniques have been developed for characterizing protein-protein interactions. One such technique is the protein-fragment complementation assay (PCA), which involves splitting a reporter protein into two fragments and fusing each fragment to one of the interacting proteins. If the proteins interact, the two fragments of the reporter protein are brought close enough to reconstitute its activity, indicating the interaction. This technique can detect weak interactions and does not require prior knowledge of the interacting partners.
Another advanced technique, the fluorescence resonance energy transfer (FRET) assay, involves labeling the interacting proteins with fluorescent molecules. When the proteins interact, energy is transferred between the fluorescent molecules, resulting in a change in fluorescence intensity that can be measured. This technique allows for the real-time detection of protein-protein interactions and can be used with live cells.
Finally, surface plasmon resonance (SPR) is an advanced technique used to study protein-protein interactions in real-time. SPR involves immobilizing one of the proteins on a sensor chip and measuring any changes in refractive index as the other protein interacts with it. This technique can measure kinetic and thermodynamic parameters of the interaction, providing detailed information about the binding mechanism.
Comparison
While all of these techniques can be used to study protein-protein interactions, there are some key differences between traditional and advanced techniques. Traditional techniques, such as Co-IP and pull-down assays, have a lower throughput and require a prior knowledge of one of the interacting partners. On the other hand, advanced techniques, such as PCA, FRET, and SPR, do not require prior knowledge of the interacting partners and can detect weak interactions. Additionally, some, such as FRET, can be used in live cells, while others, such as SPR, can provide real-time information about the binding mechanism.
References
- Tarassov, K., Messier, V., Landry, C. R., Radinovic, S., Serna Molina, M. M., Shames, I., Malitskaya, Y., Vogel, J., Bussey, H., Michnick, S. W. (2008). An in vivo map of the yeast protein interactome. Science, 320(5882):1465-1470. doi: 10.1126/science.1153878.
- Guha, U., Chaerkady, R., Marimuthu, A., Patterson, A. S., Kashyap, M. K., Harsha, H. C., Sato, M., Bader, J. S. (2008). Comparisons of tyrosine phosphorylated proteins in cells expressing lung cancer-specific alleles of EGFR and KRAS. Proceedings of the National Academy of Sciences of the United States of America, 105(37):14112-14117. doi: 10.1073/pnas.0805633105.
- Guo, Y., Chi, X., Wei, D., Shu, S., Wang, R., Wang, T., Su, Y., Du, X., Yang, S. (2018). Magnetic capture of protein-protein interactions by proximity ligation. Nature Communications, 9(1):5255. doi: 10.1038/s41467-018-07710-0.