A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions.

Nitrocellulose-filter binding is a powerful technique commonly used to study protein-nucleic acid interactions; however, its utility in quantitative studies is often compromised by its lack of precision. To improve precision and accuracy, we have introduced two modifications to the traditional technique: the use of a 96-well dot-blot apparatus and the addition of a DEAE membrane beneath the nitrocellulose membrane. Using the dot-blot apparatus, an entire triplicate set of data spanning 20-24 titrant concentrations can be collected on a single 4.5 x 5 inch sheet of nitrocellulose, obviating the need to manipulate separate filters for each titration point. The entire titration can then be quantitated simultaneously with direct two-dimensional beta-emission imaging technology. The DEAE second membrane traps all DNA that does not bind to the nitrocellulose, enabling a direct determination of the total amount of DNA filtered. This measurement improves precision by allowing the amount of DNA retained by the nitrocellulose to be normalized against the total amount of DNA filtered. The DEAE membrane also permits a more accurate quantitation of filter-retention efficiency and nonspecific background retention based on free DNA rather than total DNA filtered. The general approach and methods of analysis to obtain equilibrium binding isotherms are discussed, using as examples our studies of the Escherichia coli Rep protein, a helicase, and its interactions with short oligodeoxynucleotides. IMAGES:

硝酸纤维素膜结合法（Nitrocellulose-filter binding）是一种常用于研究蛋白质-核酸相互作用的高效技术，但其因精度不足，在定量研究中的应用常受限制。为提升精度与准确性，我们对传统技术做出两处改良：采用96孔斑点印迹装置（96-well dot-blot apparatus），并在硝酸纤维素膜下方增设DEAE膜（DEAE membrane）。借助该斑点印迹装置，单张4.5×5英寸的硝酸纤维素膜即可完成涵盖20至24种滴定剂浓度的完整三组重复数据采集，无需为每个滴定点单独处理滤膜。随后可通过直接二维β放射成像技术，对整个滴定过程同步进行定量分析。增设的DEAE膜可捕获所有未结合至硝酸纤维素膜的DNA，从而直接测定过滤总DNA量。该测定方式可通过将硝酸纤维素膜保留的DNA量与过滤总DNA量进行归一化处理，提升实验精度。此外，DEAE膜还可基于游离DNA（而非过滤总DNA），更准确地定量滤膜保留效率与非特异性背景保留量。本文以大肠杆菌Rep蛋白（一种解旋酶）与短寡脱氧核苷酸的相互作用研究为例，阐述了获取平衡结合等温线的通用方法与分析流程。图像资料：