![]() The activation of transcription factors and binding to target genes results in gene activation. Using electrophoretic mobility shift assays (EMSA) or ELISA, you can assess further downstream effects, including the activation of transcription factors (e.g., NF-kB) in the nucleus of a cell as well as the DNA regions they bind. If you want to investigate receptor-ligand binding, a biosensor assay might be the way to go. ![]() Eventually, the receptors dissociate from the ligands, and the rate of this dissociation is measured in real time using biosensors. Soluble T cell receptors are then exposed to these bound ligands and binding can occur. In a biosensor assay the ligand of interest is fixed to a gold-plated surface. Most biosensor assays use optical biosensors and the underlying principle is the same as SPR. When Should You Use a Biosensor Assay?īiosensor assays are useful in immunology and cell biology to measure the rate of association and dissociation of ligands to their specific antigen receptors. A SPR biosensor that allows measurement of the interaction at different temperatures facilitates a thermodynamic analysis of the interaction of interest. The real beauty of SPR, however, is that it allows you to take quantitative measurements of the affinity, thermodynamics, and kinetics of the interaction in real time. SPR has become the gold standard for kinetic and affinity determination, and is the underlying principle behind many color-based biosensor chip applications. This allows you to bypass the extra work involved in engineering fluorescent tags and avoid the hassle of using radioactivity. In contrast to Co-Ip, SPR does not require you to label your protein of interest. SPR is a very attractive technique for a number of reasons. Studying protein-protein (such as receptor-ligand interactions), small molecule-protein, or cell-protein interactions can be done using surface plasmon resonance (SPR). When Should You Use Surface Plasmon Resonance (SPR)? Investigating protein-protein interactions requires a more complex form of blotting called co-immunoprecipitation (Co-IP). ![]() Activation of proteins often involves phosphorylation of specific residues, and you can detect such phosphorylations with a good primary antibody. Standard western blotting can detect the activation of proteins involved in a signaling pathway. This article will help you choose the right method according to your specific needs. The most appropriate method depends on what aspect of the response you are investigating and what questions you are asking as a researcher. The cellular response to the external stimulus e.g., the activation/deactivation of intracellular signaling pathways and/or the secretion of proteins is often the research goal, and there are a number of different methods that you can use to analyze such responses. Stimulation of cells/tissue with a given stimulus (e.g., a cytokine) is a common experimental setup in any cell biology lab.
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