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Figure 7. Assaying transport by filtration, the “quick and easy” method. A radioactively tagged transport substrate is added to RSO vesicles (50-100 µL) in the presence or absence of an energy source (see below). At a given time, the vesicles are separated from the surrounding medium by rapid dilution and vacuum (VAC) filtration through filters with a pore diameter smaller than the diameter of the vesicles. The rate of filtration must be very rapid in order to trap the accumulated substrate. The method is reasonably quantitative for charged and neutral substrates (but accumulation of weak acids, in particular, is drastically underestimated because of passive permeability in the protonated state).