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生物素和光裂解链接器所附 rna 对高分子配合物的单步纯化

JoVE Journal
Biochemistry

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JoVE Journal Biochemistry

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

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生物素和光裂解链接器所附 rna 对高分子配合物的单步纯化

DOI:

10.3791/58697-v

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08:12 min

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January 03, 2019

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Aleksandra Skrajna, Xiao-Cui Yang, Zbigniew Dominski²

¹Integrative Program for Biological and Genome Sciences,University of North Carolina at Chapel Hill, ²Department of Biochemistry and Biophysics,University of North Carolina at Chapel Hill

Chapters

00:04Title
00:51Substrate Preparation
01:28Complex Assembly
02:14Immobilization of the RNA/protein Complexes on Streptavidin Beads
04:00UV-Elution
05:08Sample Analysis by Silver Staining Followed by Mass Spectrometry
05:59Results: Analysis of UV-eluted Complexes
07:36Conclusion

Summary

Automatic Translation

采用肉毒分蛋白策略纯化的 rna* 蛋白复合物在变性条件下被洗脱, 以不适合进一步纯化和功能分析的形式进行溶液处理。在这里, 我们描述了这种策略的修改, 利用光裂解链接器在 rna 和温和的紫外线洗脱步骤, 产生本地和全功能的 rna 蛋白复合物。

Tags

RNA Protein Complexes Single-step Purification Biotin Photo-cleavable Linker Mass Spectrometry T7 Generated RNA Adaptor Oligonucleotide Mouse Nuclear Extract EDTA Streptavidin Agarose Beads Binding Buffer Centrifugation Tube Rotation

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