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Abstract：
The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications, such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion-loaded nickel-nitrilotriacetic acid-based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion-loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion-loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.
摘要：
多组氨酸标签(His-tag)是生命科学中最常用的蛋白质标签之一。传统上,His标记蛋白的检测依赖于抗His抗体的免疫印迹.这种方法对于某些应用来说是费力的，如蛋白质纯化，时间和简单性至关重要。His标签也可以通过与荧光团缀合的金属离子负载的镍-次氮基三乙酸螯合剂头直接检测，这是免疫印迹的一种方便的替代方法。通常,这种螯合剂头与绿色或红色荧光团共轭，其检测需要专门的激励源和检测系统。这里,我们证明，运行后染色是通过PAGE和印迹膜中负载金属离子和荧光标记的螯合剂头检测His标签的理想选择。此外,通过比较不同螯合剂头的性能，我们展示了在扩散受限的环境中，微观亲和常数的差异如何转化为检测极限的宏观差异，如PAGE。根据这些结果,我们设计了一个简单的方法，叫做UVHis-PAGE，使用金属离子负载和荧光标记的螯合剂头检测PAGE和印迹膜中的His标记蛋白。我们的方法使用紫外线透射器作为激发源，结果可以用肉眼目视检查。
