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

Erica Gerace1, Danesh Moazed1

  • 1Department of Cell Biology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.

Methods in Enzymology
|June 23, 2015
PubMed
Summary

This study details a protocol for isolating protein complexes using the tandem affinity purification (TAP) tag system. This method enables rapid purification and analysis of protein complexes, including their composition and modifications.

Keywords:
Affinity purificationCalmodulin binding (CAM-B)Calmodulin elution (CAM-E)IgG-SepharoseLysis bufferProtease cleavageProtein lysatesTAP tagsTEV cleavage (TEV-C)

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Protein complexes play crucial roles in cellular processes.
  • Understanding protein complex composition and interactions is vital for biological research.
  • Existing purification methods may have limitations in efficiency or scope.

Purpose of the Study:

  • To present a robust protocol for the isolation and analysis of protein complexes.
  • To demonstrate the utility of the tandem affinity purification (TAP) tag system for this purpose.
  • To facilitate downstream analysis of purified protein complexes.

Main Methods:

  • Utilizing a TAP tag, which consists of two protein A domains and a calmodulin binding peptide, separated by a TEV cleavage site.
  • Purifying proteins fused to the TAP tag through a two-step affinity chromatography process.
  • Employing specific elution strategies to release the purified protein complexes.

Main Results:

  • Successful isolation of protein complexes from biological samples.
  • Demonstration of the TAP tag system's efficiency in capturing target proteins and their interactors.
  • Obtained purified complexes suitable for various downstream analyses.

Conclusions:

  • The described TAP tag protocol provides a powerful and versatile method for protein complex isolation.
  • This technique allows for comprehensive analysis of protein complex stoichiometry, posttranslational modifications, structure, and function.
  • The protocol is valuable for advancing research in molecular biology and proteomics.