Abstract
This protocol describes the extraction of RNA-binding proteins (RBPs) from cell lysates. In order to pull down target RBPs, 5-bromo-UTP (BrUTP)-incorporated RNA probes are used, which are generated by in vitro transcription. The schematic diagram (Flowchart) with procedure is indicated (Figure1 and Figure 2).Figure 1. Schematic diagram of procedure (A-H). Flow chart of experimental procedure is indicated at A-H.Figure 2. Linearization of plasmids by restricted enzyme. The plasmid is cut at restriction sites adjacent to its cloning element.
Keywords: RNA-protein interactions, 5-bromo-UTP-incorporated RNA, Mass spectrometry, Pull-down assay
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This work was supported by funds of the Japanese Science and Technology Agency and the Japanese Ministry of Education, Culture, Sports, Science and Technology for integrated promotion of social system reform and research and development, and the Kishimoto foundation.
References
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