Marchalant Yannick, Brownjohn Philip W, Bonnet Amandine, Kleffmann Torsten, Ashton John C
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).
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)
J Histochem Cytochem. 2014 Jun;62(6):395-404. doi: 10.1369/0022155414530995. Epub 2014 Mar 26.
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.
用于检测和定量膜整合蛋白，特别是G蛋白偶联受体（GPCR）的基于抗体的方法，一直受到一抗特异性问题的困扰。在本报告中，我们使用免疫印迹结合质谱法，研究了用于大麻素CB2受体（一种GPCR）的最常用商业抗体之一。通过这种方式，我们能够开发出强大的阴性对照和新型阳性对照。通过这样做，我们能够证明，一种抗体有可能对感兴趣的蛋白质（在这种情况下是CB2）敏感，但仍然与其他蛋白质发生交叉反应，因此缺乏特异性。具体而言，我们能够使用蛋白质印迹法结合质谱法明确鉴定过表达细胞系中的CB2蛋白。这表明仅用阳性对照验证抗体的常见做法不足以确保抗体的可靠性。此外，我们的工作首次开发了一种使用质谱法的无标记蛋白质检测方法，经过进一步完善，该方法可以明确鉴定天然组织中的CB2受体蛋白。
