Stojanovic Tamara, Orlova Michaela, Sialana Fernando J, Höger Harald, Stuchlik Stanislav, Milenkovic Ivan, Aradska Jana, Lubec Gert
Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, 1090, Vienna, Austria.
Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia.
Amino Acids. 2017 Jun;49(6):1101-1109. doi: 10.1007/s00726-017-2408-3. Epub 2017 Mar 18.
Dopamine receptors 1 and 2 (DRD1, DRD2) are essential for signaling in the brain for a multitude of brain functions. Previous work using several antibodies against these receptors is abundant but only the minority of antibodies used have been validated and, therefore, the results of these studies remain uncertain. Herein, antibodies against DRD1 (Merck Millipore AB1765P, Santa Cruz Biotechnology sc-14001, Sigma Aldrich D2944, Alomone Labs ADR-001) and DRD2 (Abcam ab21218, Merck Millipore AB5084P, Santa Cruz Biotechnology sc-5303) have been tested using western blotting and immunohistochemistry on mouse striatum (wild type and corresponding knock-out mice) and when specific, they were further evaluated on rat and human striatum. Moreover, a DRD1 antibody and a DRD2 antibody that were found specific in our tests were used for immunoprecipitation with subsequent mass spectrometrical identification of the immunoprecipitate. Two out of nine antibodies (anti DRD1 Sigma Aldrich D2944 and anti DRD2 Merck Millipore AB5084P) against the abovementioned dopamine receptors were specific for DRD1 and DRD2 as evaluated by western blotting and immunohistochemistry and the immunoprecipitate indeed contained DRD1 and DRD2 as revealed by mass spectrometry. The observed findings may question the use of so far non-validated antibodies against the abovementioned dopamine receptors. Own observations may be valuable for the interpretation of previous results and the design of future studies using dopamine receptors DRD1 or DRD2.
多巴胺受体1和2（DRD1、DRD2）对于大脑中多种脑功能的信号传导至关重要。此前使用多种针对这些受体的抗体开展的研究数量众多，但仅有少数使用的抗体经过了验证，因此，这些研究的结果仍不确定。在此，我们使用蛋白质免疫印迹法和免疫组织化学方法，在小鼠纹状体（野生型和相应的基因敲除小鼠）上对针对DRD1的抗体（默克密理博AB1765P、圣克鲁斯生物技术sc-14001、西格玛奥德里奇D2944、阿拉莫内实验室ADR-001）和针对DRD2的抗体（艾博抗ab21218、默克密理博AB5084P、圣克鲁斯生物技术sc-5303）进行了检测，当抗体具有特异性时，我们又在大鼠和人类纹状体上进一步评估。此外，我们还使用在测试中发现具有特异性的一种DRD1抗体和一种DRD2抗体进行免疫沉淀，随后通过质谱鉴定免疫沉淀物。通过蛋白质免疫印迹法和免疫组织化学评估，针对上述多巴胺受体的九种抗体中有两种（抗DRD1西格玛奥德里奇D2944和抗DRD2默克密理博AB5084P）对DRD1和DRD2具有特异性，并且质谱分析显示免疫沉淀物中确实含有DRD1和DRD2。这些观察结果可能会让人质疑目前使用的针对上述多巴胺受体的未经验证的抗体。我们自己的观察结果可能有助于解释先前的研究结果，并为未来使用多巴胺受体DRD1或DRD2开展的研究设计提供参考。
