Comparative study of commercially available and homemade anti-VAMP7 antibodies using CRISPR/Cas9-depleted HeLa cells and VAMP7 knockout mice

Agathe Verraes; Beatrice Cholley; Thierry Galli; Sebastien Nola

doi:10.12688/f1000research.15707.2

Comparative study of commercially available and homemade anti-VAMP7 antibodies using CRISPR/Cas9-depleted HeLa cells and VAMP7 knockout mice

F1000Res. 2018 Oct 16:7:1649. doi: 10.12688/f1000research.15707.2. eCollection 2018.

Authors

Agathe Verraes^#¹, Beatrice Cholley^#², Thierry Galli^{1

2}, Sebastien Nola^{1

2}

Affiliations

¹ 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.
² Membrane Traffic in Healthy & Diseased Brain, Institute of Psychiatry and Neuroscience of Paris, INSERM U1266, Université Paris Descartes, Sorbonne Paris-Cité, 75014, France.

^# Contributed equally.

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: CRISPR/Cas9; KO; SNARE; VAMP7; immunoprecipitation; monoclonal; polyclonal.

Publication types

Research Support, Non-U.S. Gov't

Grants and funding

Work in our group was funded by grants from Association Française contre les Myopathies (Research Grant 16612), the French National Research Agency (NeuroImmunoSynapse ANR-13-BSV2-0018-02; MetDePaDi ANR-16-CE16-0012), the Ecole des Neurosciences de Paris (ENP), the Fondation pour la Recherche Médicale (FRM), Prix Coup d’Elan pour la recherche française of the Fondation Bettencourt Schueller, awards of the Association Robert Debré pour la Recherche Médicale to T.G.