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. 2000 Jan 24;148(2):247–252. doi: 10.1083/jcb.148.2.247

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Among proteins of the SNARE complex, only free syntaxin interacts with nSec1. nSec1 (nSEC1) and all SNARE complex components, syntaxin1A (SYN1), SNAP-25 (SN25), and VAMP2, were purified as monomeric species by affinity chromatography. SYN1/SNAP-25 (binary) and nSec1/SYN1 complexes were assembled and further purified by size-exclusion chromatography. The bead-binding assays were performed by prebinding purified GST-tagged nSec1 or nSec1/SYN1 onto glutathione-agarose beads. The beads were then incubated with the indicated components overnight at 4°C. After washing, sample buffer was added to the beads and the proteins were separated on a 16% SDS-polyacrylamide gel. Molecular mass markers are indicated on the left in kD. Apparent molecular weights: monomeric nSec1, 70 kD; SYN1, 31 kD; SN25, 25 kD; and VAMP2, 10 kD.