Validating Antibodies to the Cannabinoid CB2 Receptor

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.

Neuronal expression of CB2 cannabinoid receptor mRNAs in the mouse hippocampus

“…7A), which corresponded to monomers and homodimers of CB1Rs, respectively (Wager-Miller et al, 2002), implying that GPCRs might be successfully isolated under our conditions. Our data are in agreement with the previous reports of the low specificity of anti-CB2R antibodies currently available from major vendors (Ashton, 2012; Baek et al, 2013; Cecyre et al, 2014; Marchalant et al, 2014). …”

“…As reported (Baek et al, 2013; Cecyre et al, 2014; Marchalant et al, 2014), our validation of anti-CB2R antibodies also failed. Most antibodies we used displayed multiple bands in Western blots and all the bands at any molecular weight similarly appeared in both C57BL/6J and CB2R KO mouse samples.…”

“…This characterization requires a specific anti-CB2R antibody. Although anti-CB2R antibodies have been successfully (i.e., with KO controls) used to detect CB2Rs in some studies (Van Sickle et al, 2005; Zhang et al, 2014a), other reports have demonstrated that the specificity of many anti-CB2R antibodies is not reliable at least in some types of tissues (Ashton, 2012; Baek et al, 2013; Cecyre et al, 2014; Marchalant et al, 2014). Thus, we first tested the specificity of anti-CB2R antibodies obtained from major commercial vendors using hippocampal samples.…”

“…Contradictory results have also been reported regarding whether CB2Rs are expressed in the brain stem (Derbenev et al, 2004; Van Sickle et al, 2005; Baek et al, 2008). In fact, skepticism about the specificity of many anti-CB2R antibodies has been expressed, demonstrating the current limitation in immunological detection of CB2Rs (Ashton, 2012; Baek et al, 2013; Cecyre et al, 2014; Marchalant et al, 2014). …”

An endocannabinoid system is present in the mouse olfactory epithelium but does not modulate olfaction

“…Thus, we suspect that the positive CB2 receptor immunoblot was due to the presence of CB2 receptors on immune cells located in the lamina propria. Unfortunately, we were unable to confirm this using a CB2 receptor antibody specifically for immunohistochemistry due to problems with the specificity (Baek et al, 2013; Cecyre et al, 2014; Marchalant et al, 2014). …”