Open Peer Review Any reports and responses or comments on the article can be found at the end of the article. ANTIBODY VALIDATION ARTICLE Comparative study of commercially available and homemade anti-VAMP7 antibodies using CRISPR/Cas9-depleted HeLa cells and VAMP7 knockout mice [version 2; peer review: 2 approved] Agathe Verraes , Beatrice Cholley , Thierry Galli , Sebastien Nola 1,2 Membrane Traffic in Health and Disease, INSERM ERL U950, Univ Paris Diderot, Sorbonne Paris Cité, Institut Jacques Monod, CNRS UMR 7592, Paris, France, 75013, France Membrane Traffic in Healthy & Diseased Brain, Institute of Psychiatry and Neuroscience of Paris, INSERM U1266, Université Paris Descartes, Sorbonne Paris-Cité, 75014, France Equal contributors 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 use visual scoring for immunocytochemistry staining to determine the extent of the antibodies’ specificity. Thus, 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. Keywords VAMP7, SNARE, monoclonal, polyclonal, CRISPR/Cas9, KO, immunoprecipitation This article is included in the Antibody Validations gateway. 1* 2* 1,2 1,2 1 2 * Referee Status: Invited Referees version 2 published 07 Feb 2019 version 1 published 16 Oct 2018 1 2 report report report , University of Alison H. Banham Oxford, UK 1 , University of Guelph, Marc G. Coppolino Canada , University of Olivia Grafinger Guelph, Canada 2 16 Oct 2018, :1649 ( First published: 7 ) https://doi.org/10.12688/f1000research.15707.1 07 Feb 2019, :1649 ( Latest published: 7 ) https://doi.org/10.12688/f1000research.15707.2 v2 Page 1 of 17 F1000Research 2019, 7:1649 Last updated: 20 MAR 2019