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After warming, more labelled v-SNARE remains full length: lane 2 is approximately 80–85% of the reference signal, compared with about 40–45% in lane 1. Formation of a protected SNARE complex provides a consistent explanation: the susceptible part of v-SNARE is less accessible after complex assembly.

Protection is not itself a unique readout of membrane fusion. Some protein may face the vesicle lumen, and SNARE complexes can protect protein without proving that the two aqueous compartments have merged. The inference depends on comparing these alternatives with the detergent and partner-free controls.

In lane 3, detergent removes the lipid bilayer barrier while both partner preparations are present. Their binding regions can then encounter one another even if they originally faced inward or were on poorly apposed lipid bilayers. The strong remaining full-length signal is consistent with extensive formation of protected SNARE complexes.

Thus detergent does not prevent partner association under the displayed conditions; it removes the topological restriction on that association. This lane is the normalization reference, not a direct measurement that 100% of all initially supplied protein is active. The weak signal in the detergent-treated partner-free control also argues against detergent simply preventing all cleavage.

Cold preincubation can accumulate docked, fusion-ready intermediates. On warming, that population proceeds promptly to lipid mixing, whereas newly combined vesicles must first encounter, dock and assemble productive SNARE complexes. This explains the faster initial rise and larger signal at the displayed endpoint without requiring a different total amount of fluorescent lipid.

The original reconstitution experiments likewise distinguish docking from subsequent membrane fusion.^[ref]