The presynaptic membrane fusion is mediated by a protein set called SNARE (Soluble NSF Attachment protein REceptor) proteins. SNARE proteins form a ternary SNARE-complex that comprises minimal machinery for membrane fusion; the complex consists of three SNARE proteins: Syntaxin 1, SNAP-25 and Vamp 2, also called Synaptobrevin 2. The SNARE complex is a four-helix coiled coil with four SNARE motifs; two come from SNARE-25 and one each from Syntaxin 1 and Vamp 2. It is believed that a regulatory protein Complexin binds tightly to the SNARE complex and stabilizes the complex, preventing it from driving toward fusion. However, the detailed mechanism of fusion clamping is still unclear. In our work, we constructed a singlemolecule lipid-mixing assay on a supported lipid bilayer to investigate the role of Complexin 1, one of the important regulatory proteins. Moreover, we found that Synaptotagmin 1, a calcium sensor for Ca2+-triggered fusion, plays a role along with Complexin in fusion clamping. Furthermore, the supported lipid bilayer was also incorporated into a photo-bleaching assay to investigate the role of various lipids on Syntaxin 1 clustering.