Application of tag protein covalently bonded to substrate in CLIP

Abstract

The invention discloses an application of a tag protein covalently bonded to a substrate in CLIP (cross-linking immunoprecipitation), and provides an application of a tag fusion protein covalently bonded to the substrate in a CLIP technology in a denaturant purifying manner. An RNA library used for sequencing is obtained by using the tag fusion protein covalently bonded to the substrate in a protein denaturant washing manner, and a result proves that an experiment result published in previous literatures can be well repeated. Steps of membrane transfer and gel extraction for recovering fragments in traditional CLIP processes are omitted. Labels covalently bonded to the substrate and a specific binder immobilized on a medium form covalent bonds, so strong washing of magnetic beads, which cannot be directly used previously, is added to the experiment operating steps, thereby the operation is simple, the operating time is saved, the CLIP process is optimized, and the loss possibly causedin the operating process is reduced.

Description

technical field [0001] The invention belongs to the fields of RNA biology and molecular biology, and relates to the application of a label protein capable of covalently binding a substrate in a CLIP (cross-linking immunoprecipitation, ultraviolet cross-linking immunoprecipitation) experiment, specifically relating to CLIP technology , using a fusion protein with a tag that can covalently bind to the substrate, the protein-RNA covalently cross-linked complex was purified by washing with denaturing reagents, thereby omitting SDS-PAGE (SDS-polyacrylamide) electrophoresis, cellulose acetate Steps such as prime transfer to membrane or isotope labeling, etc., finally obtain a cDNA library that can be used for sequencing. Background technique [0002] The molecular biology approach to study RNA-binding proteins and the RNA they bind to, in a nutshell, is by purifying complexes of RNA and RNA-binding proteins. The complex can be purified by either precipitating RNA or RNA-binding p...

AI Technical Summary

Problems solved by technology

[0011] Although CLIP technology has been improved many times, SDS-PAGE electrophoresis and cellulose acetate transfer are required in the process of operation.

These two steps are time-consuming, require high operation, and samples are easy to lose

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