Stimulation of cells/tissue with a given stimulus (e.g., a cytokine) is a common experimental setup in any cell biology lab. 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. The most appropriate method depends on what aspect of the response you are investigating and what questions you are asking as a researcher. This article will help you choose the right method according to your specific needs.
When Should You Use a Western Blot?
Standard western blotting can detect the activation of proteins involved in a signaling pathway. Activation of proteins often involves phosphorylation of specific residues, and you can detect such phosphorylations with a good primary antibody. Investigating protein-protein interactions requires a more complex form of blotting called co-immunoprecipitation (Co-IP).
When Should You Use Surface Plasmon Resonance (SPR)?
Studying protein-protein (such as receptor-ligand interactions), small molecule-protein, or cell-protein interactions can be done using surface plasmon resonance (SPR). SPR is a very attractive technique for a number of reasons. In contrast to Co-Ip, SPR does not require you to label your protein of interest. This allows you to bypass the extra work involved in engineering fluorescent tags and avoid the hassle of using radioactivity. 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. A SPR biosensor that allows measurement of the interaction at different temperatures facilitates a thermodynamic analysis of the interaction of interest.
When Should You Use a Biosensor Assay?
Biosensor assays are useful in immunology and cell biology to measure the rate of association and dissociation of ligands to their specific antigen receptors. Most biosensor assays use optical biosensors and the underlying principle is the same as SPR. In a biosensor assay the ligand of interest is fixed to a gold-plated surface. Soluble T cell receptors are then exposed to these bound ligands and binding can occur. Eventually, the receptors dissociate from the ligands, and the rate of this dissociation is measured in real time using biosensors. If you want to investigate receptor-ligand binding, a biosensor assay might be the way to go.
When Should You Use EMSA or ELISA?
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.
When Should You Use PCR?
The activation of transcription factors and binding to target genes results in gene activation. PCR and real-time PCR analysis methods allows you to accurately detect the expression of activated genes.
When Should You Definitely Use ELISA?
Or you can look outward from the cell. You can use ELISA analysis of cell culture supernatants to investigate extracellular protein secretion as a result of specific gene activation or silencing.
Moyes DL, Naglik JR. (2012) Analysis of host-cell responses by immunoblotting, ELISA, and real-time PCR. Methods Mol Biol. 845:345–60.
Originally published in 2014. Updated and republished in May 2017.Image Credit: ILRI