By Jiping Xiao, Clare Bergson (auth.), Nadine Kabbani (eds.)
Dopamine, a catecholamine transmitter, performs a couple of very important physiological roles within the mind and physique, and, in recent times, experiences at the position of dopamine in disorder have opened new avenues of study and discovery. In Dopamine: tools and Protocols, specialists and key figures in the box supply designated protocols on top methods within the examine of dopamine inside organic platforms. Divided into sections on cellular/biochemical, imaging, genetics, and electrophysiology, this collections contains protocols for bioluminescence and fluorescence imaging, receptor immunoprecipitation and proteomic research, construction and characterization of a mouse version of Parkinson’s sickness, genuine time dimension of dopamine within the mind, and modeling sign transduction in silico. Written within the hugely profitable Methods in Molecular Biology™ sequence structure, chapters comprise introductions to their respective matters, lists of fabrics and reagents, step by step, quite simply reproducible laboratory protocols, and tips about troubleshooting and averting recognized pitfalls.
Authoritative and handy, Dopamine: equipment and Protocols serves as a terrific advisor for college kids and specialists alike, in addition to for somebody attracted to exploring the huge and the most important box of dopamine research.
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Additional info for Dopamine: Methods and Protocols
M. Ventura et al. 2. Wash columns with 6 mL of water each and discard; elute columns three times with 3 mL of water and collect. Determine radioactivity in 150 μL of the fractions by scintillation spectroscopy; also determine radioactivity in 50 μL of [3H]-cAMP tracer solution. Calculate the % recovery of [3H]-cAMP in tubes; Select the tubes with highest recovery to measure cAMP. 3. cAMP Determination in Samples 1. 0, at 4°C, for 90 min; add H2O to complete the volume of the reaction to 200 μL (see Note 17).
Medium should be changed every 3 days. 3. After approximately 10 days, cell cultures are treated with 4 mM ascorbic acid for 2 h to eliminate neurons (13). M. Ventura et al. Fig. 2. Dopamine has been investigated as a developmental signal in the retinal tissue or cultures prepared in many different ways. (a) Mixed neuron–glial cells (prepared in high density, with ~20 × 106 cells) is ideal for functional assays measuring receptor mediated second messenger shifts, binding or western blot analysis.
Remove supernatant and add 1/10 vol. of 3 M NaOAc. 9. Add 2 vol. of 100% ethanol and incubate for 20 min at −70°C. 10. Centrifuge at 54,300 × g at 4°C. 11. Wash pellet with 70% ethanol. 12. Discard supernatant and add 20 μL DEPC-treated water. 13. Determine the concentration of RNA in samples using a spectrophotometer. 3. First Strand Synthesis of cDNA 1. Incubate RNA preparation for 10 min at 65°C to denature eventual double strand segments in RNA. 2. Dilute RNA to 1 μg/20 μL with DEPC-treated water.