Brūmele Baiba, Serova Evgeniia, Lupp Aleksandra, Suija Mihkel, Mutso Margit, Kurg Reet
Institute of Technology, University of Tartu, 50411 Tartu, Estonia.
Antibodies (Basel). 2024 Apr 22;13(2):33. doi: 10.3390/antib13020033.
Primary antibodies are one of the main tools used in molecular biology research. However, the often-occurring cross-reactivity of primary antibodies complicates accurate data analysis. Our results show that three commercial polyclonal antibodies raised against N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) strongly cross-react with endogenous and recombinant mitosis-associated protein Aurora kinase A (AURKA). The cross-reactivity was verified through immunofluorescence, immunoblot, and immunoprecipitation assays combined with mass spectrometry. N6AMT1 and AURKA are evolutionarily conserved proteins that are vital for cellular processes. Both proteins share the motif ENNPEE, which is unique to only these two proteins. We suggest that N6AMT1 antibodies recognise this motif in N6AMT1 and AURKA proteins and exhibit an example of "specific" non-specificity. This serves as an example of the importance of controls and critical data interpretation in molecular biology research.
一抗是分子生物学研究中使用的主要工具之一。然而，一抗经常出现的交叉反应会使准确的数据分析变得复杂。我们的结果表明，三种针对N-6腺嘌呤特异性DNA甲基转移酶1（N6AMT1）制备的商业多克隆抗体与内源性和重组有丝分裂相关蛋白极光激酶A（AURKA）强烈交叉反应。通过免疫荧光、免疫印迹和免疫沉淀分析结合质谱法验证了交叉反应。N6AMT1和AURKA是对细胞过程至关重要的进化保守蛋白。这两种蛋白都有ENNPEE基序，该基序仅为这两种蛋白所特有。我们认为，N6AMT1抗体识别N6AMT1和AURKA蛋白中的这一基序，并展示了“特异性”非特异性的一个例子。这说明了对照和关键数据解释在分子生物学研究中的重要性。
