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

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

Journal of neuroscience methods (2020-03-08)
Pedro F Esteban, Daniel Garcia-Ovejero, Beatriz Paniagua-Torija, Rafael Moreno-Luna, Luis F Arredondo, Andreas Zimmer, Angel Arevalo-Martin, Eduardo Molina-Holgado
摘要

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.

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Sigma-Aldrich
聚-D-赖氨酸 氢溴酸盐, mol wt 70,000-150,000, lyophilized powder, γ-irradiated, BioReagent, suitable for cell culture
Sigma-Aldrich
丽春红S染液(零售包装) 溶液, BioReagent, suitable for electrophoresis, 0.1 % (w/v) in 5% acetic acid
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胞嘧啶 β-D-呋喃阿拉伯糖苷, crystalline, ≥90% (HPLC)
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胆酸钠 水合物, BioXtra, ≥99%