In vitro assay of the interaction between Rnc1 protein and Pmp1 mRNA by affinity capillary electrophoresis with a carboxylated capillary
In vitro assay of the interaction between Rnc1 protein and Pmp1 mRNA by affinity capillary electrophoresis with a carboxylated capillary
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DOI：
10.1016/j.jpba.2010.07.009
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发表时间：
2010-12-15
影响因子：
3.4
通讯作者：
Sugiura, Reiko
中科院分区：
文献类型：
--
作者：
Taga, Atsushi;Satoh, Ryosuke;Sugiura, Reiko
采用亲和毛细管电泳（ACE）技术研究了RNA相互作用蛋白Rnc1与Pmp1 mRNA的相互作用。在ACE实验之前，用核糖核酸酶B （RNase B）和牛血清白蛋白（BSA）等模型蛋白研究了三种毛细管（未处理的熔融二氧化硅毛细管、聚乙烯-聚丙烯酸（PB-PAA）双层涂层毛细管和共价修饰的羧化毛细管）的柱性能。使用未处理的熔融二氧化硅和PB-PAA双层涂层毛细管，两者的蛋白峰均呈宽尾状。而在羧化毛细管中，蛋白峰尖锐对称，且迁移次数可重复（RSD < 0.4%）。此外，每次运行前用1 M氯化钠溶液预处理毛细管内壁，人血清中的蛋白也出现尖锐的峰，且其重复性保持在较高水平。使用羧化毛细管，用含有不同浓度Pmp1感觉mRNA的背景电解质，通过ACE分析Rnc1蛋白。mRNA浓度的增加延迟了蛋白的迁移时间。但随着Pmp1反义mRNA而非Pmp1义mRNA的增加，Rnc1蛋白的迁移时间保持不变。通过1/（迁移时移）与1/（Pmp1义mRNA浓度）的对比，得到一条直线（r=0.987），估计Rnc1蛋白与Pmp1义mRNA的关联常数为4 15 × 10(6) M（-1）。这些结果表明，用该方法可以很容易地确定以mrna为配体的蛋白质的结合常数。(C) 2010 Elsevier B.V.版权所有
The interaction between Rnc1, an RNA interactive protein, and a Pmp1 mRNA was investigated by affinity capillary electrophoresis (ACE). Prior to the ACE experiments, the column performances of three capillaries (an untreated fused silica capillary, a polybrene-polyacrylic acid (PB-PAA) double layer coating capillary, and a carboxylated capillary with a covalent modification) were studied with model proteins including ribonuclease B (RNase B) and bovine serum albumin (BSA) Using an untreated fused silica and a PB-PAA double layer coating capillaries, both of the protein peaks were broad and tailing. However, using a carboxylated capillary, the protein peaks were sharp and symmetric, and migration times were repeatable (RSD < 0 4%) Further, the proteins in human serum also gave sharp peaks and its repeatability was kept at a high level by pre-treatment of a capillary inner wall with 1 M sodium chloride solution before each run. An Rnc1 protein was analyzed by ACE with background electrolytes containing various concentrations of Pmp1 sense mRNA using a carboxylated capillary. Increase in the concentration of the mRNA was found to delay the migration time of the protein. But the migration time of the protein was kept constant with increasing Pmp1 anti-sense mRNA instead of Pmp1 sense mRNA A straight line (r=0.987) was obtained by plotting 1/(migration time shift) versus 1/(Pmp1 sense mRNA concentration) and the association constant of Rnc1 protein with Pmp1 sense mRNA could be estimated to be 4 15 x 10(6) M(-1). These results suggest that the association constants of proteins with mRNAs as ligands were easily determined by the proposed method. (C) 2010 Elsevier B.V. All rights reserved.