title: Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.
abstract: Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.
authors.affiliation: Aix-Marseille University, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie, Marseille, France (YM,AB)Department of Pharmacology and Toxicology (PWB,JCA)Centre for Protein Research, Biochemistry Department, University of Otago, Dunedin, New Zealand (TK).
authors.forename: Yannick
authors.initials: Y
authors.lastname: Marchalant
authors.affiliation: Aix-Marseille University, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie, Marseille, France (YM,AB)Department of Pharmacology and Toxicology (PWB,JCA)Centre for Protein Research, Biochemistry Department, University of Otago, Dunedin, New Zealand (TK) phil.brownjohn@gmail.com.
authors.forename: Philip W
authors.initials: PW
authors.lastname: Brownjohn
authors.affiliation: Aix-Marseille University, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie, Marseille, France (YM,AB)Department of Pharmacology and Toxicology (PWB,JCA)Centre for Protein Research, Biochemistry Department, University of Otago, Dunedin, New Zealand (TK).
authors.forename: Amandine
authors.initials: A
authors.lastname: Bonnet
authors.affiliation: Aix-Marseille University, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie, Marseille, France (YM,AB)Department of Pharmacology and Toxicology (PWB,JCA)Centre for Protein Research, Biochemistry Department, University of Otago, Dunedin, New Zealand (TK).
authors.forename: Torsten
authors.initials: T
authors.lastname: Kleffmann
authors.affiliation: Aix-Marseille University, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie, Marseille, France (YM,AB)Department of Pharmacology and Toxicology (PWB,JCA)Centre for Protein Research, Biochemistry Department, University of Otago, Dunedin, New Zealand (TK).
authors.forename: John C
authors.initials: JC
authors.lastname: Ashton
keywords: CB2; cannabinoid; immunoblot; mass spectrometry; membrane enrichment; western blot