Monthly Archives: June 2016

A Functional Assay for GPR55: Envision Protocol.

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“AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.”

http://www.ncbi.nlm.nih.gov/pubmed/27245893

The Displacement Binding Assay Using Human Cannabinoid CB2 Receptor-Transfected Cells.

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“Displacement binding assays are nonfunctional assays mostly used with the aim of determining whether a certain compound (plant-derived or synthetic) is able to bind to a specific receptor with high affinity. Here, we describe the displacement binding assay that is carried out with a radioligand and CHO (Chinese Hamster Ovarian) cells stably transfected with the human cannabinoid CB2 receptor.”

http://www.ncbi.nlm.nih.gov/pubmed/27245891

Assay of CB1 Receptor Binding.

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“Type-1 cannabinoid receptor (CB1), one of the main targets of endocannabinoids, plays a key role in several pathophysiological conditions that affect both central nervous system and peripheral tissues. Today, its biochemical identification and pharmacological characterization, as well as the screening of thousands of novel ligands that might be useful for developing CB1-based therapies, are the subject of intense research. Among available techniques that allow the analysis of CB1 binding activity, radioligand-based assays represent one of the best, fast, and reliable methods.Here, we describe radioligand binding methods standardized in our laboratory to assess CB1 binding in both tissues and cultured cells. We also report a high-throughput radioligand binding assay that allows to evaluate efficacy and potency of different compounds, which might represent the basis for the development of new drugs that target CB1 receptor-dependent human diseases.”

http://www.ncbi.nlm.nih.gov/pubmed/27245890

Need for Methods to Investigate Endocannabinoid Signaling.

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“Endocannabinoids (eCBs) are endogenous lipids able to activate cannabinoid receptors, the primary molecular targets of the cannabis (Cannabis sativa) active principle Δ(9)-tetrahydrocannabinol. During the last 20 years, several N-acylethanolamines and acylesters have been shown to act as eCBs, and a complex array of receptors, metabolic enzymes, and transporters (that altogether form the so-called eCB system) has been shown to finely tune their manifold biological activities. It appears now urgent to develop methods and protocols that allow to assay in a specific and quantitative manner the distinct components of the eCB system, and that can properly localize them within the cell. A brief overview of eCBs and of the proteins that bind, transport, and metabolize these lipids is presented here, in order to put in a better perspective the relevance of methodologies that help to disclose molecular details of eCB signaling in health and disease. Proper methodological approaches form also the basis for a more rationale and effective drug design and therapeutic strategy to combat human disorders.”

http://www.ncbi.nlm.nih.gov/pubmed/27245886