Revisiting CB cannabinoid receptor detection and the exploration of its interacting partners.

BACKGROUND: Cannabinoid receptor 1 (CB) 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 CB by WB and immunoprecipitation (IP) as a first step towards the study of CB interactome.

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

RESULTS: Sample heating above 65 °C greatly impaired CB 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 CB by WB and, mostly, for IP.

COMPARISON WITH EXISTING METHOD(S): We obtain consistent and specific CB detection by WB and IP using four different commercial antibodies and KO tissue for an accurate CB identification. We clarify the identification of the receptor in complex samples compared with the diverse and unclear results obtained using standard WB methods.

CONCLUSIONS: We establish experimental guidelines for the detection of CB by WB and the study of CB interacting proteins by IP. We propose a new interpretation of CB 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 CB.

Copyright © 2020 Elsevier B.V. All rights reserved.

PMID: 32145227