By Mauro Maccarrone
This quantity encompasses all significant methodologies to interrogate endocannabinoid platforms (ECS) and endocannabinoids (eCBs) signaling. With expanding curiosity in the direction of the manifold actions of eCBs, this publication discusses the chemical, biochemical, and molecular organic assays, and job of detailed parts of the ECS. those comprise membrane, nuclear receptors, biosynthetic and hydrolytic enzymes, and membrane transporters and oxidative enzymes. Written within the hugely profitable Methods in Molecular Biology series layout, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and tips about troubleshooting and fending off recognized pitfalls.
Timely and leading edge, Endocannabinoid Signaling: tools and Protocols is a useful source and should aid chemists, drug designers, biochemists, molecular biologists, telephone biologists, pharmacologists, and (electro) physiologists navigate the mare magnum of endocannabinoid research.
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Extra resources for Endocannabinoid Signaling: Methods and Protocols (Methods in Molecular Biology)
In the protocol presented here, the dried extract is reconstituted in 100 μl of ACN; for most autosamplers, this means that vials with inserts have to be used. The right type of vial and the appropriate injection needle height have to be selected in the LC systems settings. Assay of ω-3 Endocannabinoids 39 6. Mass spectrometry is an advanced analytical technique that allows characterizing and/or quantifying levels of molecules in a certain matrix. Different types of MS machines are available, which all have their particular strengths, weaknesses, and area of application.
We use the eCB levels determined in a reference. 19. We typically transfer 30 μl of the extracts into LC vials or well plates for analysis. The leftover 20 μl of extracts are immediately frozen and stored at −20 °C for an additional analytical replicate if necessary, or can be used for additional analysis in another ionization mode, if polarity switching is not available on the mass spectrometer. 20. The parameters and conditions for LC/MRM are valid for the lab equipment mentioned here. For other MS, LC instruments, or other LC column provider type all these settings have to be optimized accordingly.
Ensure that all samples have reached room temperature before proceeding to the ACN extraction step. This may take 30–60 min. 2. While the plasma samples are thawing, prepare the deuterated standard spiking work solution. 3. 0 ml of plasma to a clean 15 ml tube. 4. 0 ml plasma. The ACN will precipitate all proteins and highly lipophilic compounds, while keeping the endocannabinoids in solution. The ACN will completely mix with the plasma. While adding this solution to the plasma, keep the tube on a vortexer running at a gentle pace.