Van Oss C J, Good R J, Chaudhury M K
J Chromatogr. 1987 Mar 27;391(1):53-65. doi: 10.1016/s0021-9673(01)94304-3.
The transfer of DNA fractions from hydrophilic gels to nitrocellulose membranes (Southern blotting) which was soon followed by the description of an analogous procedure for RNA (Northern blotting), and somewhat later for proteins (Western blotting), has rapidly become an important separation and characterization method in molecular biology, genetic engineering, and immunological detection. Surface tension measurements have shown that the interfacial attraction between DNA and cellulose esters (-delta G132) in aqueous media can be considerable. The weaker binding energy of proteins to cellulose nitrate and to cellulose acetate may be compared to hydrophobic interaction chromatography, as on account of the somewhat lower [-delta G132] values, it often is necessary to "fix" them more tightly onto nitrocellulose by using high salt concentrations. The binding energy of RNA to both cellulose esters also is rather low. In addition to the effect of high ionic strength, the effect of adding methanol, and the effects of denaturation, heating and drying on the energy of attachment of the biopolymers to cellulose esters, have been studied. Cationized nylon membranes have been advocated recently, especially for electrophoretic transfer of nucleic acids (in which process high salt concentrations cannot easily be used). With positively charged nylon membranes, the attachment mainly occurs through the electrostatic attraction between the strongly negatively charged nucleic acids (or proteins) and the positively charged membrane. Also, more apolar membranes (of polyvinyl difluoride) have been proposed, which manifest a strong interfacial (hydrophobic) attraction to all the above biopolymers (regardless of their electrostatic charge). However, with these two novel membrane types it is no longer possible to exploit the large difference in binding energy between DNA and RNA, which makes cellulose nitrate membranes so uniquely suited for RNA-DNA hybridization assays.
将DNA片段从亲水性凝胶转移至硝酸纤维素膜上（Southern印迹法），随后不久便出现了类似的RNA转移方法（Northern印迹法），再后来又有了蛋白质转移方法（Western印迹法），这些方法迅速成为分子生物学、基因工程及免疫检测中重要的分离和鉴定手段。表面张力测量结果表明，在水性介质中，DNA与纤维素酯之间的界面吸引力（-ΔG132）可能相当可观。蛋白质与硝酸纤维素及醋酸纤维素的结合能较弱，这可与疏水相互作用色谱法相比较，因为由于[-ΔG132]值略低，通常需要使用高盐浓度将它们更紧密地“固定”在硝酸纤维素上。RNA与这两种纤维素酯的结合能也相当低。除了高离子强度的影响外，还研究了添加甲醇的影响以及变性、加热和干燥对生物聚合物与纤维素酯结合能的影响。最近有人提倡使用阳离子化尼龙膜，特别是用于核酸的电泳转移（在此过程中不能轻易使用高盐浓度）。对于带正电荷的尼龙膜，附着主要通过带强负电荷的核酸（或蛋白质）与带正电荷的膜之间的静电吸引发生。此外，还提出了更具非极性的膜（聚偏二氟乙烯膜），它对上述所有生物聚合物都表现出强烈的界面（疏水）吸引力（无论其静电荷如何）。然而，对于这两种新型膜，不再能够利用DNA和RNA在结合能上的巨大差异，而正是这种差异使得硝酸纤维素膜非常适合用于RNA-DNA杂交分析。
