Flag-biotin Clip-seq
Flag-biotin Clip-seq - Related Videos
09:54
RNA-Seq
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2023
Among different methods to evaluate gene expression, the high-throughput sequencing of RNA, or RNA-seq. is particularly attractive, as it can be performed and analyzed without relying on prior available genomic information. During RNA-seq, RNA isolated from samples of interest is used to generate a DNA library, which is then amplified and sequenced. Ultimately, RNA-seq can determine which genes are expressed, the levels of their expression, and the presence of any previously unknown transcripts.
08:46
Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification
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Cited by 1 •
2020
Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.
05:16
Assessing the Internalization of Target Surface Proteins Using a Biotin Derivative
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2025
This video demonstrates a method to assess target protein internalization in the mouse cortical astrocytes using biotinylation, followed by cell lysis, streptavidin-based protein extraction, denaturation, and Western blot analysis to confirm successful internalization.
05:07
Rup (RNA-seq Usability Assessment Pipeline) - Quality Control for Bulk RNA-seq Experiments in Eukaryotes
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2025
This protocol allows initial quality control for RNA-seq experiments for wet-lab biologists with limited bioinformatics experience.
12:24
PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
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Cited by 267 •
2010
RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.
FAQs
Ultraviolet irradiation is the capture-enabling step: it covalently links the tagged RNA-binding protein to RNA that is nearby at the time of exposure. Because the protein-RNA association is chemically fixed, the linked material can proceed through affinity purification rather than relying only on a noncovalent interaction. This provides the physical basis for recovering associated RNA.
FLAG and biotin serve as complementary affinity handles on the tagged protein. Their presence allows the crosslinked protein-RNA complexes to be selectively purified from other cellular material before the RNA is processed. These handles focus the experiment on RNA associated with the designated RNA-binding protein, strengthening the connection between recovered sequences and protein occupancy.
Sequencing provides the readout used to locate binding sites across the associated transcripts. After purification, the RNA is converted into sequencing libraries, and the resulting sequences are analyzed to identify where binding occurs. The workflow therefore converts biochemical enrichment of RNA-protein complexes into an interaction map that shows the positions of RNA-binding protein association.
At a high level, the workflow moves from crosslinking to selective recovery and then to readout. Ultraviolet irradiation first fixes protein-RNA contacts, while FLAG and biotin affinity handles support purification of the complexes. The associated RNA is then converted into sequencing libraries, and analysis of those libraries identifies protein-associated binding sites for interpretation.
Flag-biotin CLIP-seq can identify which transcripts are associated with a particular RNA-binding protein, rather than inferring regulation only from changes in RNA abundance. The resulting interaction map can be examined alongside transcript processing, stability, localization, or translation. This helps researchers connect binding events with specific post-transcriptional regulatory mechanisms.
In genetics, the maps help examine how RNA-protein interactions contribute to phenotypes. If binding patterns change in a developmental condition or disease context, the data can provide a mechanistic link between altered post-transcriptional regulation and the phenotype. They can also help investigate whether disease-associated genetic variation affects regulatory interactions involving the RNA-binding protein.