Polyacrylamide gel electrophoresis (PAGE) and immunoblotting (Western blotting) are the most common methods in life science. In conjunction with these methods, the polyhistidine-tag has proven to be a superb fusion tag for protein purification as well as specific protein detection by immunoblotting, which led to a vast amount of commercially available antibodies. Nevertheless, antibody batch-to-batch variations and nonspecific binding complicate the laborious procedure. The interaction principle applied for His-tagged protein purification by metal-affinity chromatography using N-nitrilotriacetic acid (NTA) was employed to develop small high-affinity lock-and-key molecules coupled to a fluorophore. These multivalent NTA probes allow specific detection of His-tagged proteins by fluorescence. Here, we report on HisQuick-PAGE as a fast and versatile immunoblot alternative, using such high-affinity fluorescent super-chelator probes. The procedure allows direct, fast, and ultra-sensitive in-gel detection and analysis of soluble proteins as well as intact membrane protein complexes and macromolecular ribonucleoprotein particles.

聚丙烯酰胺凝胶电泳（PAGE）和免疫印迹（Western印迹）是生命科学中最常见的方法。结合这些方法，聚组氨酸标签已被证明是用于蛋白质纯化以及通过免疫印迹检测特异性蛋白质的极佳融合标签，从而产生了大量可商购的抗体。然而，抗体的批次间差异和非特异性结合使繁琐的过程变得复杂。通过使用N-亚硝基三乙酸（NTA）的金属亲和色谱法，将相互作用原理应用于His-tagged蛋白的纯化，以开发与荧光团偶联的小的高亲和力锁键分子。这些多价NTA探针可通过荧光特异性检测His标记的蛋白。这里，我们使用这种高亲和力的荧光超级螯合剂探针在HisQuick-PAGE上报告了一种快速，通用的免疫印迹替代方法。该程序可直接，快速和超灵敏地在凝胶中检测和分析可溶性蛋白以及完整的膜蛋白复合物和大分子核糖核蛋白颗粒。