Specific detection of CB1 receptors;: cannabinoid CB1 receptor antibodies are not all created equal!
Specific detection of CB1 receptors;: cannabinoid CB1 receptor antibodies are not all created equal!
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DOI：
10.1016/j.jneumeth.2008.02.014
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发表时间：
2008-06-15
影响因子：
3
通讯作者：
Graham, E. Scott
中科院分区：
文献类型：
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作者：
Grimsey, Natasha L.;Goodfellow, Catherine E.;Graham, E. Scott
内源性大麻素CB 1受体蛋白在神经组织和细胞的研究依赖于高度特异性抗体的可用性。我们已经测试了一系列CB 1抗体检测脑中内源性受体以及使用免疫学方法的组合转染到HEK-293细胞中的血凝素（HA）标记的受体的能力。几种CB 1抗体在小鼠脑中的初步比较显示，通过放射自显影术，配体结合的染色模式存在实质性差异。然后针对表达HA标记的大鼠和人CB 1受体的HEK细胞免疫细胞化学地测试抗体。在这种情况下，测试的商业抗体都不能检测到受体。然后使用来自HEK细胞的裂解物和啮齿动物脑匀浆通过Western印迹筛选所有抗体。同样，没有一种市售抗体在转染的细胞系或脑匀浆中检测到正确分子量的蛋白质，尽管所有抗体都识别脑组织中的多种蛋白质。我们得出结论，我们测试的市售抗体未能检测到在HEK细胞中大量表达的CB 1受体或脑切片或匀浆中的天然受体。因此，在使用前必须全面验证这些CB 1抗体对特定应用的特异性。(c)2008 Elsevier B. V.保留所有权利。
The study of endogenous cannabinoid CB1 receptor proteins in neuronal tissues and cells relies on the availability of highly specific antibodies. We have tested the ability of a series of CB1 antibodies to detect endogenous receptors in brain as well as hemagglutinin (HA)-tagged receptors transfected into HEK-293 cells using a combination of immunological methods. An initial comparison of several CB1 antibodies in mouse brain revealed substantial differences in staining pattern to ligand binding by autoradiography. Antibodies were then tested immunocytochemically against HEK cells expressing HA-tagged rat and human CB1 receptors. None of the commercial antibodies tested were able to detect the receptor in this context. All antibodies were then screened by Western blotting using lysates from the HEK cells and rodent brain homogenates. Again, none of the commercially available antibodies detected proteins of the correct molecular weight in transfected cell lines or brain homogenates, although all recognized multiple proteins in brain tissues. We conclude that the commercially available antibodies we tested failed to detect CB1 receptors abundantly expressed in HEK cells or native receptors in brain slices or homogenates. As such, comprehensive validation of the specificity of these CB1 antibodies for a particular application is essential before use. (c) 2008 Elsevier B.V. All rights reserved.