Section on Functional Neuroanatomy, Laboratory of Cellular & Molecular Regulation, National Institute of Mental Health, NIH, Bethesda, MD 20892, USA.
J Neuroinflammation. 2011 Oct 14;8:141. doi: 10.1186/1742-2094-8-141.
The characterization and cellular localization of transcription factors like NF-κB requires the use of antibodies for western blots and immunohistochemistry. However, if target protein levels are low and the antibodies not well characterized, false positive data can result. In studies of NF-κB activity in the CNS, antibodies detecting NF-κB proteins have been used to support the finding that NF-κB is constitutively active in neurons, and activity levels are further increased by neurotoxic treatments, glutamate stimulation, or elevated synaptic activity. The specificity of the antibodies used was analyzed in this study.
Selectivity and nonselectivity of commonly used commercial and non-commercial p50 and p65 antibodies were demonstrated in western blot assays conducted in tissues from mutant gene knockout mice lacking the target proteins.
A few antibodies for p50 and p65 each mark a single band at the appropriate molecular weight in gels containing proteins from wildtype tissue, and this band is absent in proteins from knockout tissues. Several antibodies mark proteins that are present in knockout tissues, indicating that they are nonspecific. These include antibodies raised against the peptide sequence containing the nuclear localization signals of p65 (MAB3026; Chemicon) and p50 (sc-114; Santa Cruz). Some antibodies that recognize target proteins at the correct molecular weight still fail in western blot analysis because they also mark additional proteins and inconsistently so. We show that the criterion for validation by use of blocking peptides can still fail the test of specificity, as demonstrated for several antibodies raised against p65 phosphorylated at serine 276. Finally, even antibodies that show specificity in western blots produce nonspecific neuronal staining by immunohistochemistry.
We note that many of the findings in the literature about neuronal NF-κB are based on data garnered with antibodies that are not selective for the NF-κB subunit proteins p65 and p50. The data urge caution in interpreting studies of neuronal NF-κB activity in the brain.
转录因子（如 NF-κB）的特征和细胞定位需要使用抗体进行 Western blot 和免疫组织化学分析。然而，如果靶蛋白水平较低且抗体未得到很好的表征，则可能会产生假阳性数据。在中枢神经系统中 NF-κB 活性的研究中，已使用检测 NF-κB 蛋白的抗体来支持 NF-κB 在神经元中持续激活的发现，并且神经毒性处理、谷氨酸刺激或升高的突触活性会进一步增加其活性水平。本研究分析了所使用抗体的特异性。
在缺乏靶蛋白的突变基因敲除小鼠组织的 Western blot 检测中，证明了常用商业和非商业 p50 和 p65 抗体的选择性和非选择性。
少数 p50 和 p65 抗体在包含野生型组织蛋白的凝胶中标记分子量适当的单个条带，而在敲除组织的蛋白中不存在该条带。一些抗体标记存在于敲除组织中的蛋白，表明它们是非特异性的。这包括针对 p65（MAB3026；Chemicon）和 p50（sc-114；Santa Cruz）核定位信号肽序列的抗体。一些识别靶蛋白的抗体在 Western blot 分析中仍然失败，因为它们还标记其他蛋白，而且结果不一致。我们表明，使用阻断肽进行验证的标准仍然可能无法通过特异性测试，这在针对丝氨酸 276 磷酸化的 p65 产生的几种抗体中得到证明。最后，即使在 Western blot 中显示特异性的抗体，通过免疫组织化学也会产生非特异性神经元染色。
我们注意到，文献中关于神经元 NF-κB 的许多发现都是基于使用对 NF-κB 亚基蛋白 p65 和 p50 没有选择性的抗体获得的数据。这些数据在解释大脑中神经元 NF-κB 活性的研究时需要谨慎。
