Figure 6

Two-helical SNARE proteins offer binary immobilization system. (A) Schematic showing fusions of syntaxin to the N-terminus of brevin, referred as NanoLock (NL) in this work. NL is designed to interact with SNAP25. (B) Coomassie-stained gel showing that NL and SNAP25 assemble into an SDS-resistant complex in a 30 min reaction. Molecular weights are indicated on the left. The asterisk (*) indicates putative off-pathway oligomeric complexes. (C) Surface plasmon resonance sensogram showing the retention of NL on the GST-SNAP25 chip. The red arrow indicates the baseline of GST-SNAP25 crosslinked to the chip surface while (1) shows the level of NL bound to GST-SNAP25 after 45 minutes. A series of washes follows with eluants which are unable to elute the immobilized NL: (2) 2 M NaCl, (3) 50 mM glycine, 500 mM NaCl, (4) 0.1% SDS, (5) 100 mM NaOH, (6) 1% SDS and (7) 100 mM Phosphoric acid.