Vinson C R, LaMarco K L, Johnson P F, Landschulz W H, McKnight S L
Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210.
Genes Dev. 1988 Jul;2(7):801-6. doi: 10.1101/gad.2.7.801.
We have used a recombinant bacteriophage that expresses the DNA-binding domain of C/EBP to optimize conditions for a screening technique that may facilitate the cloning of genes that encode sequence-specific DNA-binding proteins. The method relies on the expression of cDNA inserts in bacteriophage lambda gt11. Fusion protein adsorbed onto nitrocellulose filters is probed with radioactive, double-stranded DNA as a ligand. Two procedures greatly increase the level of binding between ligand and recombinant fusion protein. First, nitrocellulose filters are processed through a denaturation/renaturation regimen using 6 M guanidine hydrochloride. Second, synthetic DNA corresponding to the specific binding site is catenated extensively using DNA ligase. The combination of these procedures leads to remarkably strong detection signals. Specific DNA-binding signals can be detected on duplicate filters, and filters can be washed and reused by repeating the cycle of denaturation/renaturation.
我们使用了一种表达C/EBP DNA结合结构域的重组噬菌体，来优化一种筛选技术的条件，该技术可能有助于克隆编码序列特异性DNA结合蛋白的基因。该方法依赖于cDNA插入片段在噬菌体λgt11中的表达。吸附在硝酸纤维素滤膜上的融合蛋白用放射性双链DNA作为配体进行检测。有两个步骤可大大提高配体与重组融合蛋白之间的结合水平。首先，硝酸纤维素滤膜用6M盐酸胍进行变性/复性处理。其次，使用DNA连接酶将对应于特异性结合位点的合成DNA大量连接。这些步骤的组合可产生非常强的检测信号。在重复的滤膜上可检测到特异性DNA结合信号，并且通过重复变性/复性循环，滤膜可洗涤并重复使用。
