Tandem Affinity Purification
Tandem Affinity 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.
10:02
Identification of Protein Interacting Partners Using Tandem Affinity Purification
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Cited by 20 •
2012
Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.
06:34
Tandem Affinity Purification Assay to Study Protein-Protein Interactions
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2025
This video demonstrates tandem affinity purification — a technique to purify protein complexes from eukaryotic cells to study protein-protein interaction. The complex contains two proteins labeled with different epitope tags. Upon the purification of the complex using two resins with an affinity for the two different tags in a sequential manner, the presence of both proteins in the elute confirms the interaction between the two.
14:47
Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry
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Cited by 5 •
2016
MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.
10:36
Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method
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Cited by 4 •
2016
The Tandem Affinity Purification (TAP) method has been used extensively to isolate native complexes from cellular extract, primarily eukaryotic, for proteomics. Here, we present a TAP method protocol optimized for purification of native complexes for structural studies.
FAQs
The two selections increase purification specificity beyond a single capture step. A tagged target complex first binds one immobilized ligand, undergoes gentle elution, and then encounters a second affinity matrix. Proteins that do not remain associated with the target or that fail to bind the second ligand are less likely to remain in the final preparation, improving enrichment of the complex.
The dual affinity tag provides two distinct binding opportunities for the same expressed target protein. Each tag interacts with its corresponding immobilized ligand during a separate selection, while an intervening protease cleavage site may permit removal of the tag. This design helps retain associated partners under relatively native conditions while adding a second layer of selectivity.
Relatively native purification conditions can help preserve associations between the target protein and interacting partners during enrichment. The resulting material may therefore reveal complex composition rather than only the presence of the tagged protein. This is particularly useful when the goal is to examine protein-protein interactions or connect complexes with cellular pathways.
The workflow begins by expressing the target protein with a dual affinity tag. The resulting material is applied to the first immobilized ligand, and the captured complex is gently eluted. That eluate is transferred to a second affinity matrix for another selection; if included, protease treatment can remove the tag between these stages.
A successful setup requires an expressed, dual-tagged target protein, a first immobilized ligand, a second affinity matrix, and potentially a protease cleavage step. Gentle elution is important between selections because the objective is to recover the target together with associated partners. The purified fraction can then be prepared for biochemical analysis.
Tandem Affinity Purification is appropriate when researchers need enriched protein complexes and information about their associated partners, rather than only an isolated target protein. Its sequential selections provide added specificity, while relatively native handling supports interaction studies. In biology, the recovered material can help investigate complex composition and relationships within cellular pathways.
The final purified material can be examined by immunoblotting, mass spectrometry, or other biochemical techniques. These analyses can confirm or characterize the enriched target and its associated proteins, depending on the experimental objective. Together, the purification and downstream measurements provide a route from complex isolation to investigation of protein interactions and composition.