Application of tag protein covalently bonded to substrate in CLIP

A technology of tagging proteins and covalent binding, applied in the application field, can solve the problems of high operation requirements, easy loss of samples, and long time consumption.

Active Publication Date: 2018-03-23
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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  • Application of tag protein covalently bonded to substrate in CLIP
  • Application of tag protein covalently bonded to substrate in CLIP
  • Application of tag protein covalently bonded to substrate in CLIP

Examples

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Embodiment 1

[0062] Example 1. CLIP experiment carried out in HEK293 cell line with Halo-tagged PTB protein

[0063] In this example, the application of the GoldCLIP technology provided by the present invention in RNA-binding proteins will be studied by performing a CLIP experiment on the PTB protein with a Halo tag in the HEK293 cell line.

[0064] (1) Expression of Halo-PTB fusion protein in HEK293 cell line

[0065] The plasmid MSCV-NHalo-3xTev-PTB-T2-puro (Halo tag protein and PTB protein with the gene encoding the Halo-PTB fusion protein was passed through the specific recognition sequence Glu-Asn-Leu-Tyr- Phe-Gln-Gly / Ser connected, the map of the plasmid is as follows figure 2 As shown, the full sequence is shown in sequence 2 in the sequence listing, wherein the 1419-2309th position of sequence 2 encodes the Halo tag protein shown in sequence 1 in the sequence listing, and the 2322-2342th, 2349-2369th and 2376th positions -2396 encoding the specific recognition sequence of 3 tand...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/68G01N33/68G01N33/53
CPCC12Q1/68C12Q1/6869G01N33/5308G01N33/68C12Q2523/101C12Q2522/101C12Q2539/103C12Q2535/122
Inventor 俞洋顾嘉琦
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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