3.4. SNARE complex formation and DNA handle crosslinking

Run PCR to generate the DNA handles labeled with a thiol group at one old and two digoxigenin moieties at the other end (Fig. 1A).

Purify DNA handles using PCR Purification Kit.

Reduce DNA handles with 2 mM TCEP first and then remove free TCEP. Activate the thiol groups in the DNA handle with 1 mM DTDP and remove free DTDP.

Measure the DNA concentration.

Mix the purified syntaxin, SNAP-25 and VAMP2 proteins in a molar ratio of 0.8:1:1.2 lysis buffer and incubate the mixture at 4 °C overnight to form ternary SNARE complexes.

Purify the SNARE complexes: Add above reaction mixture to appropriate amount of washed Ni-NTA beads, incubate while rotating the bead solution at 4 °C for 2 hours to allow SNAP-25 bind to Ni-NTA beads through its N-terminal His tag. Both free SNAP-25 and ternary SNARE complexes bound to Ni-NTA beads, but only ternary complexes could bind to beads and be pulled.

Drain and then wash the beads with 500 μL Buffer C three times to remove TCEP and free VAMP2 and syntaxin molecules.

Elute the SNARE complex using elute Buffer C.

Measure the concentration of the eluted SNARE complexes.

Immediately crosslink the SNARE complex and the DNA handle (Fig. 1A): Mix the DTDP-treated DNA handles with the purified SNARE complexes at a molar ratio of 1:100.