Neurotransmitter Methods (Methods in Molecular Biology) by Richard C. Rayne
By Richard C. Rayne
Birkbeck collage, U.K. Bench-side spouse for researchers who search to spot, localize, or degree neurotransmitters and/or to spot websites of neurotransmitter motion. 36 individuals, 6 U.S. vast references. DNLM: Neurotransmitters-physiology.
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13. Pass the filtrates through 8 pm Millipore syringe filters by gentle application of positive pressure. Collect the filtrates in the respective, we-cold 12-mL centrifuge tubes. 14. Centrifuge the fjltrates at 1OOOgfor 20 min at 44X. 15. Gently resuspend the resulting pellets in 1 rnL of ice-cold HB using a 1-mL pipeter. 16. Centrifuge the suspensions at 1OOOgfor 10 min at 4-6”C. 17. Resuspend the pellets in 200 pL (CA,,) and 100 pL (DG) of HB. Determine the protein content using the Bio-Rad total protein assay (see Notes 3 and 4 and ref.
In such cases, Tris should not be used as a pH buffer because it interferes with the ammo acid analysis after o-phthaldlaldehyde (OPA)-denvatization. 2. , steps 7 and 8). We have given instructions in Section 2 2 for setting up three Percoll gradients and these instructions may be scaled up if additional gradients are required. For example, depending on the centrifuge rotor that 1s used, four gradients may be required (three to receive homogenate samples and one “blank” to be used as a balancer in the centrifuge) although in some rotors, three tubes may be balanced m a triangle arrangement.
These proteins are often the subject of study and, therefore, this step 1s required to avoid possible artifacts. Interestingly, EM studies show that this step effects a further elimmation of extrasynaptosomal mitochondna (not pelleted at the lower speed) from the Percoll C-band (compare Fig. 2B and 2D). 10 Because Ca2+ appears to be necessary to maintain stability of synaptosomes, the preparation is held in high-Ca2+ aCSF (physiological extracellular concentration) at the end of the isolation. 05 mM EGTA) for more than 1 h increases LDH activity, indicating increased membrane leakage (see Note 11).