Guenther R H, Gopal D H, Agris P F
Division of Biological Sciences, University of Missouri, Columbia 65211.
J Chromatogr. 1988 Jul 1;444:79-87. doi: 10.1016/s0021-9673(01)94010-5.
Anion-exchange high-performance liquid chromatography (HPLC) methods have been developed for the purification and concentration of milligram quantities of tRNA. A Waters Protein Pak DEAE 5PW 150 x 21.5 mm I.D. column was utilized for the separation of tRNA species. The chromatographic conditions chosen created non-denaturing conditions for separating the different species: 0.1 M Tris buffer (pH 7.6) at 25 degrees C, with a 0.25 M to 0.4 M sodium chloride gradient, using a 170-min gradient. The gradient form could be adjusted for optimizing purification (to over 85%) of the tRNA species of interest. The same DEAE packing in a smaller column was found to be effective for concentrating solutions of the purified tRNA. Fifty-fold concentration and recoveries above 90% have been obtained by this method. These methods were successfully applied to the purification of individual tRNA species from both Escherichia coli and yeast.
已开发出阴离子交换高效液相色谱（HPLC）方法用于纯化和浓缩毫克量的转运RNA（tRNA）。使用一根内径为21.5毫米、长150毫米的沃特世Protein Pak DEAE 5PW柱来分离tRNA种类。所选择的色谱条件为分离不同种类的tRNA创造了非变性条件：25℃下的0.1M Tris缓冲液（pH 7.6），采用0.25M至0.4M的氯化钠梯度，梯度时间为170分钟。梯度形式可进行调整以优化目标tRNA种类的纯化（纯化率超过85%）。发现在较小的柱子中使用相同的DEAE填料对于浓缩纯化后的tRNA溶液是有效的。通过这种方法已实现了50倍的浓缩，回收率高于90%。这些方法已成功应用于从大肠杆菌和酵母中纯化单个tRNA种类。
