PDF(Pubmed)
Abstract：
The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins.
摘要：
复杂溶液中重组蛋白的分析通常用蛋白质印迹中的标签特异性抗体完成。最近,我引入了无抗体的替代方案，其中标记的蛋白质直接在聚丙烯酰胺凝胶中可视化。为此，我使用蛋白质连接酶Connectase将荧光团选择性地附着到具有N末端识别序列的靶蛋白上。在这项研究中,我将这种方法扩展到包括C-末端标记的蛋白质的检测和定量。类似于N端标记方法，这种适应的程序提供了更高的速度，灵敏度提高,与西方印迹相比，信噪比有所提高。它还消除了对样本特定优化的需要，实现更一致和精确的量化，并使用免费提供的试剂。这项研究扩大了凝胶内荧光检测方法的适用性，从而促进了重组蛋白的研究。
