By Susan G. Amara (Eds.)
General Description of the Series:
Neurotransmitter Transporters specializes in biochemical, electrophysiological, pharmacological, molecular, and cellphone organic ways used to check neurotransmitter delivery platforms. The articles supply targeted descriptions of methods that are meant to permit the reader to appreciate how they're finished and to copy or adapt them for his or her personal experimental wishes. This booklet is the 1st to target equipment which were the foundation for the quick improvement of this area.
General Description of the Series:
The seriously acclaimed laboratory typical for greater than 40 years, Methods in Enzymology is among the so much hugely revered guides within the box of biochemistry. in view that 1955, every one quantity has been eagerly awaited, often consulted, and praised through researchers and reviewers alike. Now with greater than three hundred volumes (all of them nonetheless in print), the sequence includes a lot fabric nonetheless proper today--truly a vital ebook for researchers in all fields of lifestyles sciences.
* The delivery of CNS neurotransmitter transporters
* Electrophysiological, biochemical, molecular, mobile organic, pharmacological, neurochemical, and structural approaches
* either plasma and vesicular companies
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Additional info for Neurotransmitter Transporters
Lee, M. A. Hediger, and S. C. Hebert, J. Biol. Chem. 269, 17713 (1994). 5 mM (Romero and Nakhoul, unpublished data, 1995). Intracellular Na + was raised by blocking the oocytes' Na + pump with K+-free ND96 for 24 hr, thus allowing expression to be observed more easily. 19 Injecting Oocytes Oocytes are microinjected with usually 50 nl of fluid, with or without RNA. 5-mm nylon or polypropylene mesh. Oocytes are visualized under a dissecting microscope and injections are performed using micropipettes held by a micromanipulator.
Oo ~ ~ = o I I I t'N ~ o  EXPRESSION CLONING IN OOCYTES 35 is expressed at relatively high levels, as was the case for the ROMK1 K ÷ channel 14 and the eNaC Na + channel. 5-ml tubes. Typically a 5 or 10 ml, 5-20% sucrose gradient is made in a swinging bucket ultracentrifuge. The gradient can be made by placing equal volumes of the 20% and the 5% sucrose in a gradient mixer (two flat-bottom cylinders connected at the bottom by tubing and another outlet tube) with stirrers at the bottom. A capillary glass pipette is connected to the outlet tubing and placed at the bottom of the 5- or 10-ml centrifuge tube.
A time-dependent impairment of the glycine uptake is also observed after deglycosylation of the reconstituted GLYT2 protein obtained by transient expression in mammalian cells (data not shown). Total deglycosylation of the expressed protein by PNGase F also produces a [21 EXPRESSION CLONING IN OOCYTES 17 decrease in uptake activity (50%), the small differences being probably attributable to the specific glycosylation pattern of every cell type. 29 Acknowledgments The authors thank Dr. Jestis Vfizquez and Enrique Ntifiez for valuable collaboration.