Rna Protein Complex Purification
Rna Protein Complex Purification - Related Videos
11:30
Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells
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Cited by 7 •
2017
We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.
06:36
Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes
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Cited by 6 •
2017
Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.
14:58
Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry
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Cited by 11 •
2012
Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per...
11:09
Chromatin Isolation by RNA Purification (ChIRP)
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Cited by 380 •
2012
ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.
02:30
Isolation of Small Regulatory RNA–Bound Bacterial Target RNA Using Affinity Purification
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2025
Source: Mercier, N., et. al., MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria. J. Vis. Exp. (2021)This video demonstrates the isolation of MS2-tagged small regulatory RNA (sRNA) bound to its bacterial target RNA using affinity purification with a maltose-binding protein (MBP)–MS2 coat protein fusion immobilized on amylose resin.
FAQs
Non-denaturing conditions help maintain the native interactions between an RNA molecule and its associated proteins during isolation. If the complex loses its structure or dissociates, the recovered material may no longer represent the original biological assembly. Preserving these interactions is therefore essential when the goal is to examine binding partners, interaction stoichiometry, or molecular function.
Affinity capture uses a tag attached to either the RNA or protein component to selectively retain the complex on an affinity chromatography medium. Unbound molecules are removed during washing, while the retained assembly is recovered by elution. This strategy enriches the tagged complex and supports subsequent analysis of its associated components.
Salt concentration, detergent, and temperature can change whether an RNA-protein assembly remains intact during purification. Because these conditions influence complex stability, they must be considered when preserving native interactions. A preparation that maintains the assembly is more suitable for measuring interaction stoichiometry or investigating molecular function than material recovered after dissociation.
A typical workflow stabilizes the complex under non-denaturing conditions, applies the sample for affinity capture through a tagged RNA or protein, washes away unbound molecules, and elutes the retained material. The resulting preparation can then be examined by electrophoresis, mass spectrometry, or structural studies, depending on the information being sought.
Purified material can be analyzed by electrophoresis, mass spectrometry, or structural studies. These approaches provide complementary information about the recovered assembly, including its associated proteins, interaction stoichiometry, and structural features. Selecting among them allows researchers to connect the physical composition of the complex with questions about its molecular function.
In biochemistry, the method helps investigate post-transcriptional regulation and ribonucleoprotein assembly. It also supports studies of mechanisms controlling RNA stability, localization, and translation. By isolating RNA with its associated proteins rather than examining the components separately, researchers can relate molecular interactions to the broader processes that regulate RNA behavior.