By Zhizhong Z. Pan
This complete number of the main cross-disciplinary equipment and protocols utilized in present opioid learn covers themes starting from molecular and genetic innovations, to behavioral analyses of animal versions and to scientific practices. The well-practiced authors describe their most sensible molecular strategies for the cloning and expression of opioid receptors, and for the quantitative characterization in their signaling pathways, in addition to for mapping the distribution and detecting the expression degrees of opioid receptors, opioid peptides, and their messages in either mind tissues and person cells. additionally incorporated are equipment for the production of in vitro and in vivo animal types to check opioid capabilities, in addition to the scientific functions within the remedy of soreness and opioid dependancy.
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Additional info for Opioid Research. Methods and Protocols
3. Expose the labeled cells to treatments with varied times and concentrations as required in individual experiments. 4. Place the 6-well dish on a slide-warmed tray at 37°C. 5. 5 mL of pre-warmed medium containing treatment drug (or control) at 2× the desired final concentration and incubate for 5–20 min. 6. Following removal of the medium, cool the cells to 4°C by washing the cell twice with ice-cold phosphate-buffered saline (PBS). 7. Carry out all subsequent procedures at 4°C. 8. 5 mL/well of ice-cold “RIPA+” buffer to the cells.
Monitor the radioactivity reading with a handhold radioactivity moniter. Stop washing when the reading drops to approx 2000 cpm. 13. Put the membranes on a filter paper and let them air-dry. 14. Determine the radioactivity on the P81 membranes in a liquid scintillation counter. The background readings are usually between 2000–3000 cpm. 15. 2. PKC Assay 1. Wash the cells twice with PBS and sonicate in lysis buffer for 10 s on ice. 2. Centrifuge the cell lysate at 100,000g for 30 min at 4°C. Collect the supernatant and use it as cytosolic fraction (see Note 2).
207, 40–43. 38 Thakker et al. Kinase Assays 39 4 Analysis of Opioid-Induced Kinase Activation Lan Ma 1. Introduction Phosphorylation is the most important and common way of regulation of protein functions. It offers rapid and reversible regulation. Protein kinases catalyze phosphorylation of a protein and transfer the γ-phosphate of adenosine triphosphate (ATP) onto the serine, threonine, or tyrosine residue. It has been shown that stimulation of opioid receptors regulates activities of numerous protein kinases including protein kinase C (PKC), cAMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CamK II), mitogin-activated protein kinases (MAPKs), and G protein coupled receptor kinases (1–7).