Using Western Blotting for the Evaluation of Metabotropic Glutamate Receptors Within the Nervous System: Procedures and Technical Considerations

Western blotting is a highly adaptable biochemical assay that is used widely across cellular and molecular biology disciplines and has a diverse array of applications. This technique has been used for over four decades and remains a popular method for both protein detection and quantification. Identifying proteins of interest, such as metabotropic glutamatergic receptors (mGlu receptors), has proven beneficial for addressing key questions within the field. However, the promiscuity of mGlu receptor antibodies has yielded inconsistent results with high levels of nonspecific binding and background staining. In addition, mGlu receptors frequently form heterodimers or homodimers with other receptors. The overarching goal of this chapter is to discuss the theory of western blotting, applying of this technique for answering scientific questions regarding changes in mGlu receptor protein expression, and provide a standardized western blotting protocol for immunoblotting of these receptors. While other immunoblotting methods can also be used, this chapter contains general information that will aid researchers in the ability to conduct western blot experiments, especially those relating to mGlu receptor expression.