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Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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Related Experiment Video

Updated: Mar 16, 2026

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
10:36

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

Published on: July 27, 2016

10.1K

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method.

Clarisse van der Feltz1, Daniel Pomeranz Krummel2

  • 1Department of Biochemistry, Brandeis University.

Journal of Visualized Experiments : Jove
|August 9, 2016
PubMed
Summary

Optimized Tandem Affinity Purification (TAP) method successfully isolated a homogeneous ribonucleoprotein complex from yeast. This advance provides high-quality material for structural determination using electron microscopy.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • Affinity purification is crucial for isolating native protein complexes for proteomic analysis.
  • Structural and compositional heterogeneity can hinder proteomic studies but are prohibitive for structural characterization.
  • Advances in electron microscopy necessitate high-quality, homogeneous complexes for structural determination.

Purpose of the Study:

  • To optimize the Tandem Affinity Purification (TAP) method for isolating homogeneous macromolecular complexes.
  • To obtain sufficient quantity and quality of a specific ribonucleoprotein assembly for structural studies.
  • To detail modifications to the TAP method and validation strategies for complex homogeneity.

Main Methods:

  • Adaptation and optimization of the Tandem Affinity Purification (TAP) protocol.
  • Purification of an 18-subunit, ~0.8 MDa ribonucleoprotein assembly from Saccharomyces cerevisiae.
  • Assay development to evaluate compositional and structural homogeneity of the purified complex.

Main Results:

  • Successful isolation of a compositionally and structurally homogeneous ribonucleoprotein complex.
  • The optimized TAP method yielded sufficient material for electron microscopy-based structural analysis.
  • Characterization confirmed the suitability of the purified complex for negative stain and cryo-electron microscopy.

Conclusions:

  • The modified TAP method is effective for purifying homogeneous, large macromolecular assemblies.
  • This approach enables high-resolution structural determination of complex biological machinery.
  • The study provides a robust protocol for preparing samples for advanced structural biology techniques.