title: Detecting RNA-Protein Interaction Using End-Labeled Biotinylated RNA Oligonucleotides and Immunoblotting.
abstract: RNA-protein interaction can be detected by RNA pull-down and immunoblotting methods. Here, we describe a method to detect RNA-protein interaction using RNA pull down and to identify the proteins that are pulled-down by the RNA using immunoblotting. In this protocol, RNAs with specific sequences are biotinylated and immobilized onto Streptavidin beads, which are then used to pull down interacting proteins from cellular extracts. The presence of a specific protein is subsequently verified by SDS- polyacrylamide gel electrophoresis and immunoblotting with antibodies. Interactions between the SMN RNA and the PSF protein and between the caspase-2 RNA and the SRSF3 protein (SRp20) in nuclear extract prepared from HeLa cells are illustrated as examples.
authors.affiliation: School of Life Science, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju, 500-712, South Korea.
authors.forename: Xuexiu
authors.initials: X
authors.lastname: Zheng
authors.affiliation: School of Life Science, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju, 500-712, South Korea.
authors.forename: Sunghee
authors.initials: S
authors.lastname: Cho
authors.affiliation: School of Life Science, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju, 500-712, South Korea.
authors.forename: Heegyum
authors.initials: H
authors.lastname: Moon
authors.affiliation: School of Life Science, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju, 500-712, South Korea.
authors.forename: Tiing Jen
authors.initials: TJ
authors.lastname: Loh
authors.affiliation: School of Life Science, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju, 500-712, South Korea.
authors.forename: Ha Na
authors.initials: HN
authors.lastname: Jang
authors.affiliation: School of Life Science, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro (Oryong-dong), Buk-gu, Gwangju, 500-712, South Korea. Haihongshen@gist.ac.kr.
authors.forename: Haihong
authors.initials: H
authors.lastname: Shen
mesh_terms: D019921:Biotinylation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D012313:RNA / Q000032:analysis / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors / Q000032:analysis / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
chemical_list: D011506:Proteins; C491388:SRSF3 protein, human; D000068103:Serine-Arginine Splicing Factors; D012313:RNA; D019809:Streptavidin
keywords: Biotinylation; Immunoblotting; RNA binding protein; RNA pull-down; RNA–protein interaction; Spliceosome; Streptavidin