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Affinity Chromatography01:03

Affinity Chromatography

Affinity chromatography is a powerful technique extensively utilized for separating and purifying specific biomolecules from complex mixtures. It capitalizes on the highly selective binding between an analyte and its counterpart, such as antibody-antigen interactions. The counterpart is immobilized on the stationary phase, forming an affinity column. The stationary phase typically consists of solid support, such as agarose or porous glass beads, immobilizing the affinity ligand. The mobile...

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Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells
11:30

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Published on: January 26, 2017

A highly efficient multifunctional tandem affinity purification approach applicable to diverse organisms.

Hanhui Ma1, Janel R McLean, Lucy Fang-I Chao

  • 1Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

Molecular & Cellular Proteomics : MCP
|April 5, 2012
PubMed
Summary

A new multifunctional tandem affinity purification (MAP) tag allows simultaneous protein localization, binding partner identification, and modification analysis. This powerful tool enhances protein research by enabling efficient purification and visualization across organisms.

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Last Updated: May 23, 2026

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells
11:30

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Published on: January 26, 2017

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
10:32

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Published on: January 16, 2012

Identification of Protein Interacting Partners Using Tandem Affinity Purification
10:02

Identification of Protein Interacting Partners Using Tandem Affinity Purification

Published on: February 25, 2012

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Proteomics

Background:

  • Understanding protein function requires determining protein localization, binding partners, and post-translational modifications.
  • Existing protein tagging methods often lack the ability to simultaneously assess these crucial aspects.
  • There is a need for versatile tools to comprehensively study protein characteristics.

Purpose of the Study:

  • To introduce and validate a novel multifunctional tandem affinity purification (MAP) tag.
  • To demonstrate the MAP tag's capability for simultaneous protein localization, purification, and interaction analysis.
  • To showcase the MAP tag's utility in identifying protein modifications and binding partners.

Main Methods:

  • Development of the MAP tag by inserting affinity tags into an mVenus surface loop.
  • Utilizing mVenus fluorescence for protein localization and Fluorescence-Activated Cell Sorting (FACS).
  • Employing tandem affinity purification coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Main Results:

  • The MAP tag demonstrated high purification efficiency across multiple organisms and proteins.
  • mVenus fluorescence enabled effective protein localization and cell line selection.
  • LC-MS/MS analysis using the MAP tag successfully identified known and novel binding partners and modifications of Plk1 kinase.

Conclusions:

  • The MAP tag is a highly efficient and versatile tool for comprehensive protein analysis.
  • It facilitates simultaneous determination of protein localization, interactions, and modifications.
  • The MAP tag represents a significant advancement for proteomic studies and understanding protein function.