Kroe Rachel R, Laue Thomas M
Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT 06877, USA.
Anal Biochem. 2009 Jul 1;390(1):1-13. doi: 10.1016/j.ab.2008.11.033. Epub 2008 Dec 6.
Analytical ultracentrifugation is a widely used method for characterizing the solution behavior of macromolecules. However, the two commonly used detectors, absorbance and interference, impose some fundamental restrictions on the concentrations and complexity of the solutions that can be analyzed. The recent addition of a fluorescence detector for the XL-I analytical ultracentrifuge (AU-FDS) enables two different types of sedimentation experiments. First, the AU-FDS can detect picomolar concentrations of labeled solutes, allowing the characterization of very dilute solutions of macromolecules, applications we call normal use tracer sedimentation (NUTS). The great sensitivity of NUTS analysis allows the characterization of small quantities of materials and high-affinity interactions. Second, the AU-FDS allows characterization of trace quantities of labeled molecules in solutions containing high concentrations and complex mixtures of unlabeled molecules, applications we call biological on-line tracer sedimentation (BOLTS). The discrimination of BOLTS enables the size distribution of a labeled macromolecule to be determined in biological milieus such as cell lysates and serum. Examples that embody features of both NUTS and BOLTS applications are presented along with our observations on these applications.
分析超速离心法是一种广泛用于表征大分子溶液行为的方法。然而，两种常用的检测器，即吸光度检测器和干涉检测器，对可分析溶液的浓度和复杂性施加了一些基本限制。最近为XL-I分析超速离心机添加的荧光检测器（AU-FDS）可实现两种不同类型的沉降实验。首先，AU-FDS可以检测皮摩尔浓度的标记溶质，从而能够表征大分子的极稀溶液，我们将这些应用称为常规示踪剂沉降（NUTS）。NUTS分析的高灵敏度使得能够对少量材料和高亲和力相互作用进行表征。其次，AU-FDS能够对含有高浓度未标记分子和复杂混合物的溶液中的痕量标记分子进行表征，我们将这些应用称为生物在线示踪剂沉降（BOLTS）。BOLTS的分辨能力使我们能够在细胞裂解液和血清等生物环境中确定标记大分子的大小分布。本文介绍了体现NUTS和BOLTS应用特点的实例以及我们对这些应用的观察结果。
