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用碘125I标记核糖体核糖核酸

Labeling of ribosomal ribonucleic acid with iodine 125I.

作者信息

Gładek A, Mordarski M

出版信息

Arch Immunol Ther Exp (Warsz). 1983;31(4):555-63.

PMID:6200090
Abstract

RNA was labeled with iodine in the reaction catalyzed with lactoperoxidase (LP). Labeling of RNA requires additional purification of LP on Sephadex G-100 and application of RNase inhibitors. Immobilized LP shows lower activity than a soluble enzyme but still it is sufficient to label RNA to 7% IC. Immobilized enzyme is easy to remove from the post-reaction mixture and can be used many times. Optimum conditions for enzymatic labeling were as follows: concentration ratio C/KI for the soluble enzyme equal to 7.5 (for the immobilized enzyme--12). The oxidizing agent (H2O2) used in the enzymatic method of labeling at the concentrations required in the reaction (3.5 X 10(-5) M) does not cause the degradation of nucleic acids as opposed to the oxidizing agent (TlCl3) used in the chemical method. The degree of RNA degradation increases with the amount of incorporated iodine. The RNA preparations of high degree of labeling (10% iodocytosine) by means of the chemical and enzymatic methods do not differ much. At lower labeling the enzymatic method produces less degraded preparations.

摘要

在乳过氧化物酶(LP)催化的反应中,RNA用碘进行标记。RNA的标记需要在葡聚糖凝胶G - 100上对LP进行额外纯化,并使用核糖核酸酶抑制剂。固定化LP的活性低于可溶性酶,但仍足以将RNA标记至7%的碘胞嘧啶(IC)。固定化酶易于从反应后混合物中去除,并且可以多次使用。酶促标记的最佳条件如下:可溶性酶的C/KI浓度比等于7.5(固定化酶为12)。与化学方法中使用的氧化剂(TlCl3)不同,酶促标记方法中使用的氧化剂(H2O2)在反应所需浓度(3.5×10⁻⁵ M)下不会导致核酸降解。RNA降解程度随掺入碘的量增加而增加。通过化学和酶促方法制备的高标记度(10%碘胞嘧啶)的RNA制剂差异不大。在较低标记度时,酶促方法产生的降解产物较少。

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