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Tandem affinity purification
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Tandem affinity purification
Tandem affinity purification (TAP) is an immunoprecipitation-based purification technique for studying protein–protein interactions. The goal is to extract from a cell only the protein of interest, in complex with any other proteins it interacted with. TAP uses two types of agarose beads that bind to the protein of interest and that can be separated from the cell lysate by centrifugation, without disturbing, denaturing or contaminating the involved complexes. To enable the protein of interest to bind to the beads, it is tagged with a designed piece, the TAP tag.
The original TAP method involves the fusion of the TAP tag to the C-terminus of the protein under study. The TAP tag consists of three components: a calmodulin binding peptide (CBP), TEV protease cleavage site, and two Protein A domains, which bind tightly to IgG (making a TAP tag a type of epitope tag).
Many other tag/bead/eluent combinations have been proposed since the TAP principle was first published.
This tag is also known as the C-terminal TAP tag because an N-terminal version is also available. However, the method to be described assumes the use of a C-terminal tag, although the principle behind the method is still the same.
TAP tagging was invented by a research team working in the European Molecular Biology Laboratory in the late 1990s (Rigaut et al., 1999, Puig et al., 2001) and proposed as a new tool for proteome exploration. It was used by the team to characterize several protein complexes (Rigaut et al., 1999, Caspary et al. 1999, Bouveret et al., 2000, Puig et al., 2001). The first large-scale application of this technique was in 2002, in which the research team worked in collaboration with scientists of the proteomics company Cellzome to develop a visual map of the interaction of more than 230 multi-protein complexes in a yeast cell by systematically tagging the TAP tag to each protein. The first successful report of using TAP tag technology in plants came in 2004 (Rohila et al., 2004,)
There are a few methods in which the fusion protein can be introduced into the host cells. If the host is yeast, then one of the methods may be the use of plasmids that will eventually translate the fusion protein within the host. Whichever method that is being used, it is preferable to maintain expression of the fusion protein as close as possible to its natural level. Once the fusion protein is translated within the host, it will interact with other proteins, ideally in a manner unaffected by the TAP tag.
Subsequently, the tagged protein (with its binding partners) is retrieved using an affinity selection process.
The first type of bead added is coated with Immunoglobulin G, which binds to the TAP tag's outermost end. The beads, with the proteins of interest, are separated from the lysate via centrifugation. The proteins are then released from the beads by an enzyme (TEV protease) which breaks the tag at the TEV cleavage site in the middle.
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Tandem affinity purification
Tandem affinity purification (TAP) is an immunoprecipitation-based purification technique for studying protein–protein interactions. The goal is to extract from a cell only the protein of interest, in complex with any other proteins it interacted with. TAP uses two types of agarose beads that bind to the protein of interest and that can be separated from the cell lysate by centrifugation, without disturbing, denaturing or contaminating the involved complexes. To enable the protein of interest to bind to the beads, it is tagged with a designed piece, the TAP tag.
The original TAP method involves the fusion of the TAP tag to the C-terminus of the protein under study. The TAP tag consists of three components: a calmodulin binding peptide (CBP), TEV protease cleavage site, and two Protein A domains, which bind tightly to IgG (making a TAP tag a type of epitope tag).
Many other tag/bead/eluent combinations have been proposed since the TAP principle was first published.
This tag is also known as the C-terminal TAP tag because an N-terminal version is also available. However, the method to be described assumes the use of a C-terminal tag, although the principle behind the method is still the same.
TAP tagging was invented by a research team working in the European Molecular Biology Laboratory in the late 1990s (Rigaut et al., 1999, Puig et al., 2001) and proposed as a new tool for proteome exploration. It was used by the team to characterize several protein complexes (Rigaut et al., 1999, Caspary et al. 1999, Bouveret et al., 2000, Puig et al., 2001). The first large-scale application of this technique was in 2002, in which the research team worked in collaboration with scientists of the proteomics company Cellzome to develop a visual map of the interaction of more than 230 multi-protein complexes in a yeast cell by systematically tagging the TAP tag to each protein. The first successful report of using TAP tag technology in plants came in 2004 (Rohila et al., 2004,)
There are a few methods in which the fusion protein can be introduced into the host cells. If the host is yeast, then one of the methods may be the use of plasmids that will eventually translate the fusion protein within the host. Whichever method that is being used, it is preferable to maintain expression of the fusion protein as close as possible to its natural level. Once the fusion protein is translated within the host, it will interact with other proteins, ideally in a manner unaffected by the TAP tag.
Subsequently, the tagged protein (with its binding partners) is retrieved using an affinity selection process.
The first type of bead added is coated with Immunoglobulin G, which binds to the TAP tag's outermost end. The beads, with the proteins of interest, are separated from the lysate via centrifugation. The proteins are then released from the beads by an enzyme (TEV protease) which breaks the tag at the TEV cleavage site in the middle.
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