Quantitative Detection of μ Opioid Receptor: Western Blot Analyses Using µ Opioid Receptor Knockout Mice

Shinya      Kasai; Hideko      Yamamoto; Etsuko      Kamegaya; George      R. Uhl; Ichiro      Sora; Masahiko      Watanabe; Kazutaka      Ikeda

Abstract

Increasing evidence suggests that μ opioid receptor (MOP) expression is altered during the development of and withdrawal from substance dependence. Although anti-MOP antibodies have been hypothesized to be useful for estimating MOP expression levels, inconsistent MOP molecular weights (MWs) have been reported in studies using anti-MOP antibodies. In the present study, we generated a new anti-MOP antibody (N38) against the 1-38 amino acid sequence of the mouse MOP N-terminus and conducted Western blot analysis with wildtype and MOP knockout brain lysates to determine the MWs of intrinsic MOP. The N38 antibody detected migrating bands with relative MWs of 60-67 kDa in the plasma membrane fraction isolated from wildtype brain, but not from the MOP knockout brain. These migrating bands exhibited semi-linear density in the range of 3-30 μg membrane proteins/lane. The N38 antibody may be useful for quantitatively detecting MOP.

Keywords: Knockout mice, μ opioid receptor, quantification, Western blot analysis, alcohol-dependent, chronic cocaine, downregulates MOP mRNA, immunohistochemistry, MOP binding, mRNA levels, MOP proteins