Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity

Yannick Marchalant, Philip W. Brownjohn, Amandine Bonnet, Torsten Kleffmann, John C. Ashton

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

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.

Original languageEnglish
Pages (from-to)395-404
Number of pages10
JournalJournal of Histochemistry and Cytochemistry
Volume62
Issue number6
DOIs
StatePublished - Jun 2014

Keywords

  • CB2
  • cannabinoid
  • immunoblot
  • mass spectrometry
  • membrane enrichment
  • western blot

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