Related Experiment Video
Updated: May 7, 2026
10:53
Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution
Published on: January 16, 2017
RNA-binding protein immunoprecipitation from whole-cell extracts
Tino Köster1, Dorothee Staiger
1Department of Molecular Cell Physiology, Institute for Genome Research and Systems Biology, University of Bielefeld, Bielefeld, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|September 24, 2013
Summary
Researchers developed RNA immunoprecipitation to identify RNA-binding protein targets in Arabidopsis. This method helps understand gene regulation by pinpointing direct RNA interactions in vivo.
Area of Science:
- Molecular Biology
- Plant Science
- Genetics
Background:
- RNA-based regulation significantly influences the cellular transcriptome.
- RNA-binding proteins (RBPs) interact with cis-regulatory motifs in pre-mRNAs, controlling target RNA fate.
- Identifying direct in vivo targets of RBPs is crucial for understanding posttranscriptional regulatory networks.
Purpose of the Study:
- To describe a method for identifying RNA targets of RNA-binding proteins in Arabidopsis thaliana.
- To establish a technique for capturing and identifying RNA-protein interactions within living plant cells.
Main Methods:
- RNA immunoprecipitation (RIP) was performed on transgenic Arabidopsis plants expressing an RBP fused to green fluorescent protein (GFP).
- Formaldehyde was used to crosslink proteins to their interacting RNAs in vivo.
- Immunoprecipitation utilized an antibody against the GFP tag, with mock immunoprecipitation as a control.
- Coprecipitated RNAs were eluted and quantified using real-time PCR.
Main Results:
- Specific binding of RNAs to the RBP was indicated by enrichment in GFP-tagged immunoprecipitation compared to controls.
- The described method allows for the identification of direct RNA targets of RBPs in Arabidopsis.
- This technique provides a means to study posttranscriptional gene regulation in plants.
Conclusions:
- RNA immunoprecipitation is an effective method for identifying in vivo RNA targets of RNA-binding proteins in Arabidopsis.
- This approach facilitates the study of posttranscriptional regulatory networks in plants.
- Understanding RBP-RNA interactions is key to deciphering gene expression control.
More Related Videos
Related Concept Videos
01:20Immunoprecipitation
Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...
02:36Chromatin Immunoprecipitation- ChIP
Chromatin immunoprecipitation, or ChIP, is an antibody-based technique used to identify sites on DNA that bind to transcription factors of interest or histone proteins. It also helps determine the type of histone modifications such as acetylation, phosphorylation, or methylation.
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...
Types of ChIP
ChIP can be divided into two types - X-ChIP and N-ChIP. X-ChIP involves in vivo cross-linking of histones and regulatory proteins to DNA, fragmenting the DNA by sonication, and isolating the protein-DNA...