Werner R, Mühlbach H P, Guitton M C
Universität Hamburg, Institut für Allgemeine Botanik, Germany.
Biotechniques. 1995 Aug;19(2):218-22.
The detection and isolation of cDNAs of tomato proteins that are able to bind to viroid RNA molecules are described. They were found by screening of a lambda gt11 cDNA expression library using a modification of the previously established ligand-blotting procedure to detect DNA- and RNA-binding proteins. The essentials of our modifications are the use of (i) digoxigenin-labeled viroid RNA, (ii) low concentration of the labeled probes and (iii) an expression library that allows the direct isolation of cDNA clones. The analysis of various isolated clones showed that this method is reliable for RNA-ligand screening and North-Western blotting. Applied to viroid RNA, these experimental tools provide the precondition for further studies on the specificity of the isolated proteins.
本文描述了能够与类病毒RNA分子结合的番茄蛋白cDNA的检测与分离。通过对先前建立的用于检测DNA和RNA结合蛋白的配体印迹法进行改进，筛选λgt11 cDNA表达文库，从而发现了这些蛋白。我们改进方法的要点包括：（i）使用地高辛标记的类病毒RNA；（ii）低浓度的标记探针；（iii）能够直接分离cDNA克隆的表达文库。对各种分离克隆的分析表明，该方法对于RNA配体筛选和蛋白质免疫印迹分析是可靠的。将这些实验工具应用于类病毒RNA，为进一步研究分离蛋白的特异性提供了前提条件。
