 Solution

Lane 4 combines detergent with the absence of the t-SNARE preparation. The lipid bilayer no longer protects inward-facing v-SNARE, and there is no t-SNARE partner to provide complex-dependent protection. Consequently little full-length labelled protein remains. This distinguishes membrane-dependent protection from partner-dependent protection in a protease protection assay. It also helps interpret lane 3: its strong signal requires the partner rather than lipid bilayer enclosure alone.