Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.
Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.
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DOI：
10.1369/0022155414530995
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发表时间：
2014-06
期刊：
影响因子：
--
通讯作者：
Ashton JC
中科院分区：
文献类型：
--
作者：
Marchalant Y;Brownjohn PW;Bonnet A;Kleffmann T;Ashton JC
用于检测和定量膜整合蛋白，特别是G蛋白偶联受体（GPCR）的基于抗体的方法一直受到一抗特异性问题的困扰。在这份报告中，我们调查了一个最常用的商业抗体的大麻素CB 2受体，一个GPCR，使用免疫印迹结合质谱。通过这种方式，我们能够开发出强大的阴性和新型阳性对照。通过这样做，我们能够证明抗体可能对感兴趣的蛋白质敏感-在这种情况下是CB 2-但仍然与其他蛋白质交叉反应，因此缺乏特异性。具体地说，我们能够使用蛋白质印迹结合质谱法，明确地确定CB 2蛋白在过表达细胞系。这表明仅用阳性对照验证抗体的常规做法不足以确保抗体的可靠性。此外，我们的工作是第一个开发一种无标记的蛋白质检测方法，使用质谱，进一步完善，可以提供明确的识别天然组织中的CB 2受体蛋白。
Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest—in this case CB2—but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.
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影响因子：
7.3
作者：
Curto-Reyes, V.;Llames, S.;Baamonde, A.
通讯作者：
Baamonde, A.
人体免疫组织和白细胞亚群中枢和外周大麻素受体的表达
DOI：
10.1111/j.1432-1033.1995.tb20780.x
发表时间：
1995-08-15
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
GALIEGUE, S;MARY, S;CASELLAS, P
通讯作者：
CASELLAS, P
影响因子：
7
作者：
Silva, JC;Gorenstein, MV;Geromanos, SJ
通讯作者：
Geromanos, SJ
DOI：
10.1073/pnas.93.25.14440
发表时间：
1996-12-10
影响因子：
11.1
作者：
Shevchenko, A;Jensen, ON;Mann, M
通讯作者：
Mann, M
影响因子：
64.8
作者：
MUNRO, S;THOMAS, KL;ABUSHAAR, M
通讯作者：
ABUSHAAR, M