illustra MicroSpin G-50 Columns

illustra MicroSpin G-50柱

Company: Cytiva

Catalog#: 27-5330-02

Single Oligonucleotide Capture of RNA And Temperature Elution Series (SOCRATES) for Identification of RNA-binding Proteins

Allen T. Yu, Disha Aggarwal, Darryl Pappin and David L. Spector

Published online: 2022-12-20

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UV Cross-linking Assay and Competition Assay

Author:

Ying-Wen Huang and Yau-Heiu Hsu,

Date:

2012-12-20

[Abstract] UV cross-linking assay is a standard method used to detect protein-RNA interaction. This method takes advantage of UV irradiation to trigger the formation of the covalently bonded RNP (ribonucleoprotein) complex that is more stable and makes it possible to be isolated in the denaturing conditions. Briefly, ³²P-labeled RNA probe and proteins are incubated to form RNP complexes spontaneously; the mixture is then exposed to UV irradiation, followed by treatment with ribonuclease to remove RNA fragments not covalently bound to protein. The oligoribonucleotide-protein complexes are analyzed by SDS-PAGE, and the signals visualized by phosphorimaging. Competitive UV cross-linking assay is a method to determine the protein binding sites and specificity on the RNA substrate. In this ... [摘要] UV交联测定是用于检测蛋白-RNA相互作用的标准方法。该方法利用UV辐射来触发更稳定的共价结合的RNP（核糖核蛋白）复合物的形成，并且使得可以在变性条件下分离。简言之，将32 P标记的RNA探针和蛋白质自发孵育以形成RNP复合物;然后将混合物暴露于UV辐射，随后用核糖核酸酶处理以除去未与蛋白质共价结合的RNA片段。通过SDS-PAGE分析寡核糖核苷酸 - 蛋白复合物，并通过磷光成像显现信号。竞争性UV交联测定是确定蛋白质结合位点和对RNA底物的特异性的方法。在该测定中，将过量的未标记的竞争剂RNA与蛋白质预孵育，然后加入32 P标记的RNA探针。如果竞争剂RNA包含蛋白质结合区，则蛋白质和RNA探针之间的结合将竞争并且放射性结合信号将降低。

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illustra MicroSpin G-50 Columns

illustra MicroSpin G-50柱

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