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使用短生物素化RNA对RNA结合蛋白进行优化的定量下拉分析

JoVE Journal
Bioquímica

This content is Free Access.

JoVE Journal Bioquímica

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

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使用短生物素化RNA对RNA结合蛋白进行优化的定量下拉分析

DOI:

10.3791/64923-v

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07:55 min

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February 17, 2023

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Núria Crua Asensio, Stefano Rizzieri, Alessandro Cuomo, Gian Gaetano Tartaglia³, Elsa Zacco

¹Center for Human Technology,Italian Institute of Technology (IIT), ²Department of Experimental Oncology,European Institute of Oncology (IEO), ³Department of Biology and Biotechnologies ‘Charles Darwin’,Sapienza University of Rome

Capítulos

00:05Introduction
00:42Total Protein Harvest and Protein Concentration Determination
01:48Bead Preparation
03:12Bead Loading and Protein Binding on Beads
04:13Washing of Nonspecific Binders
04:53Elution of Specific Binders
05:39Results: Quantification and Validation of Protein Interactors of Specific RNA Sequences
07:24Conclusion

Summary

Tadução automática

在这里，我们提出了一种优化的体外方法，使用来自人类细胞的总蛋白质提取物、涂有生物素化RNA的链霉亲和素珠和质谱分析来发现，定量和验证特定RNA序列的蛋白质相互作用物。

Tags

Keywords: RNA-binding Proteins Pull-down Analysis Biotinylated RNA Protein Complexes HEK 293T Cells Lysis Buffer Magnetic Beads Yeast TRNA RNA Oligonucleotide

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