Western Blotting of the Endocannabinoid System

Abstract

Measuring protein levels of receptors and enzymes involved in endocannabinoid metabolism is an important step for understanding the distribution, function, and regulation of these components of the endocannabinoid system. A common approach for detecting proteins from complex biological systems is western blotting. In this chapter, we describe a general approach to western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes with a focus on detecting type 1 cannabinoid (CB₁) receptors. When this technique is carefully used, with due attention paid to the validation of the primary antibodies used, it can provide quantitative information on protein expression levels. Additional information can also be inferred from western blotting such as potential pre- and post-translational modifications (e.g., alternative splicing, phosphorylation, or glycosylation) that can be further evaluated by specific analytical techniques.

Keywords: CB1 cannabinoid receptor (CB1); Epitope tag; Fluorescent secondary antibody detection; G protein-coupled receptor (GPCR); Gel electrophoresis.