Western blot (protein immunoblot) is a widely used analytical technique in molecular biology. Utilizing the specific recognizing primary antibody, proteins immobilized on various matrix are investigated by subsequent visualization steps, for example, by the horse radish peroxidase conjugated secondary antibody incubation. Methods to improve the sensitivity in protein identification or quantification are appreciated by biochemists. Herein, we report a new strategy to amplify Western blot signals by constructing a probe with proximal labeling and IgG targeting abilities. The R118G mutation attenuated the biotin‐AMP binding affinity of the bacterial biotin ligase BirA*, offering a proximity‐dependent labeling ability, which could be used as a signal amplifier. We built a BirA*‐protein A fusion protein (BioEnhancer) that specifically binds to IgG and adds biotin tags to its proximal amine groups, enhancing the immunosignal of target proteins. In our experiments, the BioEnhancer system amplified the immunosignal by tenfold compared to the standard western blot. Additionally, our strategy could couple with other signal enhancement methods to further increase the western blot sensitivity.

中文翻译：

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基于 BirA*-protein A 融合蛋白的 BioEnhancer 可放大蛋白质印迹免疫信号

蛋白质印迹（蛋白质免疫印迹）是分子生物学中广泛使用的分析技术。利用特定的识别一抗，固定在各种基质上的蛋白质通过随后的可视化步骤进行研究，例如，通过辣根过氧化物酶缀合的二抗孵育。生物化学家赞赏提高蛋白质鉴定或定量灵敏度的方法。在此，我们报告了一种通过构建具有近端标记和 IgG 靶向能力的探针来放大蛋白质印迹信号的新策略。R118G 突变减弱了细菌生物素连接酶 BirA* 的生物素-AMP 结合亲和力，提供了邻近依赖性标记能力，可用作信号放大器。我们构建了一种 BirA*-蛋白 A 融合蛋白 (BioEnhancer)，它与 ​​IgG 特异性结合，并在其近端胺基团上添加生物素标签，增强了目标蛋白的免疫信号。在我们的实验中，与标准蛋白质印迹相比，BioEnhancer 系统将免疫信号放大了十倍。此外，我们的策略可以与其他信号增强方法结合使用，以进一步提高蛋白质印迹的灵敏度。