Biotin-Streptavidin Affinity Purification of RNA-Protein Complexes Assembled In Vitro

Shuai Hou; Lei Shi; Haixin Lei

doi:10.1007/978-1-4939-3591-8_3

Biotin-Streptavidin Affinity Purification of RNA-Protein Complexes Assembled In Vitro

Methods Mol Biol. 2016:1421:23-34. doi: 10.1007/978-1-4939-3591-8_3.

Authors

Shuai Hou¹, Lei Shi¹, Haixin Lei²

Affiliations

¹ Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, No. 9 West Section Lvshun South Road, Dalian, Liaoning, 116044, People's Republic of China.
² Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, No. 9 West Section Lvshun South Road, Dalian, Liaoning, 116044, People's Republic of China. haixinlei@dlmedu.edu.cn.

PMID: 26965254
DOI: 10.1007/978-1-4939-3591-8_3

Abstract

RNA-protein complexes are essential for the function of different RNAs, yet purification of specific RNA-protein complexes can be complicated and is a major obstacle in understanding the mechanism of regulatory RNAs. Here we present a protocol to purify RNA-protein complexes assembled in vitro based on biotin-streptavidin affinity. In vitro transcribed RNA is labeled with (32)P and biotin, ribonucleoprotein particles or RNPs are assembled by incubation of RNA in nuclear extract and fractionated using gel filtration, and RNP fractions are pooled for biotin-streptavidin affinity purification. The amount of RNA-protein complexes purified following this protocol is sufficient for mass spectrometry.

Keywords: Affinity purification; Biotin; RNA–protein complexes; Streptavidin.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biotin / chemistry*
Chromatography, Affinity / methods*
Mass Spectrometry / methods
RNA / isolation & purification
Ribonucleoproteins / isolation & purification*
Streptavidin / chemistry*

Substances

Ribonucleoproteins
RNA
Biotin
Streptavidin