研究目的
Investigating the protocol for covalently linking transiently interacting RNA-protein complexes by UV crosslinking, removing the unprotected RNA by RNase digestion, and detecting the RNA-protein complexes by SDS-PAGE analysis.
研究成果
The described protocol provides a rapid and reliable means to directly assay RNA-protein interactions and their kinetics, facilitating the identification of novel RNA-protein interactions.
研究不足
The protocol requires specific equipment like UV crosslinkers and phosphorimagers, and the use of radiolabeled materials necessitates proper biosafety protocols.
1：Experimental Design and Method Selection:
The protocol involves UV crosslinking to covalently link RNA-protein complexes, RNase digestion to remove unprotected RNA, and SDS-PAGE analysis to detect the complexes.
2：Sample Selection and Data Sources:
Uses purified proteins and cell lysates for RNA-protein binding assays.
3：List of Experimental Equipment and Materials:
Includes microcentrifuge tubes, 96-well plates, transcription kits, radiolabeled UTP, RNase inhibitors, and UV crosslinkers among others.
4：Experimental Procedures and Operational Workflow:
Detailed steps for template preparation, RNA preparation, RNA-protein binding, UV crosslinking, and separation of RNA-protein complexes.
5：Data Analysis Methods:
Phosphorimaging and X-ray film development for visualizing RNA-protein complexes.
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microcentrifuge tube
MCT-150-C
Corning, Axygen?
Used for holding samples during experiments
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Transcription Kit
MAXIscript? Kit
Thermo Fisher Scientific, Invitrogen?
Used for in vitro transcription
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α-P32 UTP
BLU007H250UC
PerkinElmer
Used as a radiolabel in RNA preparation
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96-well round-bottomed plate
650101
Greiner Bio one International
Used for holding reaction mixtures during UV crosslinking
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UV cross-linker
CL1000
UVP
Used for UV crosslinking RNA-protein complexes
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Vertical gel electrophoretic system
Mini-PROTEAN Tetra Cell
Bio-Rad Laboratories
Used for SDS-PAGE analysis
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Phosphorimager
Typhoon Trio
GE Healthcare
Used for visualizing RNA-protein complexes
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