Revisiting CB₁ cannabinoid receptor detection and the exploration of its interacting partners

Cannabinoid receptor 1 (CB₁) identification by western blot (WB) has generated a great deal of controversial data making the interpretation of the results difficult. Our purpose is to find the most adequate experimental conditions to detect CB₁ by WB and immunoprecipitation (IP) as a first step towards the study of CB₁ interactome.

We use CB₁ knockout mice tissue as negative controls and describe appropriate sample handling conditions for CB₁ detection by WB and IP from brain and cortical neuron cultures.

Sample heating above 65 °C greatly impaired CB₁ detection by WB, since it favored the formation of high molecular weight aggregates. We also show the convenience of using n-dodecyl-β-d-maltoside (DDM) as a detergent for the detection of CB₁ by WB and, mostly, for IP.

We obtain consistent and specific CB₁ detection by WB and IP using four different commercial antibodies and KO tissue for an accurate CB₁ identification. We clarify the identification of the receptor in complex samples compared with the diverse and unclear results obtained using standard WB methods.

We establish experimental guidelines for the detection of CB₁ by WB and the study of CB₁ interacting proteins by IP. We propose a new interpretation of CB₁ WB and IP data based on the folding and packing state of the protein and the detergent used. The standardization of the most advantageous conditions for coimmunoprecipitation (CoIP) would be a useful tool for the future study of the interactome of CB₁.