Abstract
VAMP7 (vesicle-associated membrane protein) belongs to the intracellular membrane fusion SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein family. In this study, we used CRISPR/Cas9 genome editing technology to generate VAMP7 knockout (KO) human HeLa cells and mouse KO brain extracts in order to test the specificity and the background of a set of commercially available and homemade anti-VAMP7 antibodies. We propose a simple profiling method to analyze western blotting and immunocytochemistry staining profiles and determine the extent of the antibodies' specificity. Using this method, we were able to rank the performance of a set of available antibodies and further showed an optimized procedure for VAMP7 immunoprecipitation, which we validated using wild-type and KO mouse brain extracts.
Highlights
Intracellular membrane fusion in the secretory and endocytic pathways relies on SNARE proteins for membrane fusion events
VAMP7 is a clostridial neurotoxininsensitive v-SNARE that belongs to the “Longin” subfamily: it encompasses an amino-terminal extension, the Longin domain, which acts as an auto-regulatory domain[1]
VAMP7 exocytosis was shown to be regulated by an integrin, FAK, and Src-dependent mechanism in developing neurons[11] and its transport to the cell periphery by VARP, Rab[21] and Kif[57], while retrograde transport depends on LRRK112
Summary
University of Guelph, This article is included in the Antibody Validations gateway. Any reports and responses or comments on the article can be found at the end of the article
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