Elsevier

Methods in Enzymology

Volume 228, 1994, Pages 451-469
Methods in Enzymology

[44] Isolation of highly purified plant plasma membranes and separation of inside-out and right-side-out vesicles

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    The obtained pellet was suspended in 5 mM BTP-MES (pH 7.5) containing 330 mM sorbitol, 5 mM KCl and 0.1 mM EDTA and loaded into a two-phase system composed of 6.2% (w/w) Dextran T500, 6.2% (w/w) polyethylene glycol 3350, 330 mM sorbitol, 5 mM BTP-MES (pH 7.5) with 5 mM KCl and 0.1 mM EDTA. After centrifugation at 500g for 5 min, the upper phase was collected, loaded on a fresh lower phase prepared as described by Larson et al. [32] and centrifuged again to increase the upper fraction purity. The upper phase was then collected and diluted five-fold in 5 mM BTP-MES (pH 7.5) with 330 mM sorbitol, 5 mM KCl and 0.1 mM EDTA.

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