Extravesicular buffer and protein buffers

The buffer commonly used for chloride efflux measurements in this laboratory is that described in Peterson and Cramer (57). The buffer composition is 10 mM DMG, 100 mM choline nitrate, 2 mM Ca(N03)2. Choline nitrate is formed from choline bicarbonate (Sigma) and nitric acid. The buffer is degassed for at least 1 h and titrated to the appropriate pH with NaOH. Other chloride-free buffers are also suitable for measurement, provided their ionic strength is similar to that used for vesicle preparation.

The protein sample should be in a buffer that is chloride free, if possible, and should also lack any compounds which would interfere with electrode response. (3-Mercaptoethanol has been found to especially perturb electrode response (S. L. Schendel and J. C. Reed, unpublished results).

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