Oehler S, Alex R, Barker A
Institut für Genetik der Universität zu Köln, Weyertal 121, Köln, 50931, Germany.
Anal Biochem. 1999 Mar 15;268(2):330-6. doi: 10.1006/abio.1998.3056.
The ability to bind to nitrocellulose is commonly accepted as being a universal property of proteins and has been widely used in many different fields of study. This property was first exploited in the study of DNA-binding proteins 30 years ago, in studies involving DNA binding by the lactose repressor (LacR) of Escherichia coli. Termed the filter-binding assay, it remains the quickest and easiest assay available for the study of protein-DNA interactions. However, the exact mechanism by which proteins bind to nitrocellulose remains uncertain. Given the supposedly universal nature of the interaction, we were surprised to notice that certain LacR variants were completely unable to bind simultaneously to DNA containing a single lac operator and nitrocellulose. Investigation of this loss of binding suggests that LacR requires a protein region that is both hydrophobic in nature and more or less unstructured, in order to bind to both nitrocellulose and DNA. In the case of wild-type, tetrameric LacR, the DNA-recognition domain that is not bound to DNA suffices. Dimeric LacR variants will only bind if they have certain C-terminal extensions. These experiments sound a cautionary note for the use of filter binding as an assay of choice, particularly in applications involving screening for the DNA-binding site of putative DNA-binding proteins.
与硝酸纤维素结合的能力通常被认为是蛋白质的一种普遍特性，并已在许多不同的研究领域中广泛应用。30年前，这种特性首次在DNA结合蛋白的研究中得到利用，当时的研究涉及大肠杆菌乳糖阻遏物（LacR）与DNA的结合。这种方法被称为滤膜结合测定法，它仍然是研究蛋白质-DNA相互作用最快速、最简单的方法。然而，蛋白质与硝酸纤维素结合的确切机制仍不确定。鉴于这种相互作用被认为具有普遍性质，我们惊讶地发现某些LacR变体完全无法同时与含有单个乳糖操纵子的DNA和硝酸纤维素结合。对这种结合丧失的研究表明，LacR需要一个本质上既疏水又或多或少无结构的蛋白质区域，以便与硝酸纤维素和DNA结合。对于野生型四聚体LacR来说，未与DNA结合的DNA识别结构域就足够了。二聚体LacR变体只有在具有某些C末端延伸时才会结合。这些实验为将滤膜结合作为首选测定方法敲响了警钟，尤其是在涉及筛选假定DNA结合蛋白的DNA结合位点的应用中。
