Figure 4.

The effect of trans streptavidin on a longer carboxy-terminal fragment with a biotin attached near the amino terminus. Before the start of the record, 240 ng of biotinylated mutant 326C/CT-L (with an amino-terminal His-tag) were added to the cis compartment. The conductance turned on at +70 mV and off at −80 mV. At the arrow, 20 μg of streptavidin were added to the trans compartment. This produced an inhibition of turn off at negative voltage, with the development of a noisy conductance. This shows that the amino-terminal biotin of this long fragment was accessible to trans streptavidin. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl₂, 1 mM EDTA, 20 mM HEPES, pH 7.2. The record was filtered at 30 Hz.