Tandem-affinity-purification, TAP

Puig O, Caspary F, Rigaut G, et al. The tandem affinity purification (TAP) method a general procedure of protein complex purification. Methods 2001 24 218-229. 

Puig, 0., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M. and Seraphin, B. (2001) The tandem affinity purification (TAP) method a general procedure of protein complex purification. Methods 24, 218-229. 

Although mass spectrometry has made significant strides in sensitivity and proteome coverage, a recent report by Weissman and co-workers indicates that significant challenges remain [72]. With the use of a complete fusion, tandem-affinity-purification (TAP)-tag library for the genome of Saccharomyces cere-visiae, protein expression levels were quantitatively determined with Western blots by chemiluminescent detection. These results were compared to MS-based (MudPIT) results [54,73], and the MS data were strongly biased toward the detection of abundant proteins. For the 75% of the proteome that is represented by proteins present at fewer than 5000 copies per cell, only 8% were... 

Gavin et al. [13] made use of a combination of Tandem-Affinity purification (TAP) and mass spectrometry to characterize multiprotein complexes in S. cerevisiae. Ho et al. [14] identified protein complexes covering about 25% of the yeast proteome using a similar procedure, with a different purification step. 

Protein-protein interactions can be detected many ways in vitro. Some of these include coimmunoprecipitation, protein pulldown assay, chemical crosslinking, fluorescence resonance energy transfer (FRET), label transfer, and tandem affinity purification (TAP). Of these, TAP is the only high-throughput method. 

Fig. 1 Schematics of tandem affinity purification (TAP). The methodology of using TAP technique for protein complex purification is outlined in 6 steps. (1) The target protein (the bait ) is co-transcribed with the tandem-arranged affinity tags to either the N- or the C-terminus of the gene (shown is the N-terminal fusion). The two affinity tags (shaded boxes 1 and 2j are separated by a protease cleavage site (blank bo)(j, from where the far-end tag would be cut off and the near-end one would be exposed to the affinity columns for a second-round purification. (2) The bait and the tags are expressed as a chimeric fusion protein. The bait would form a complex with its in vivo partners (depicted as A, B, and Q. Nonspecific association may happen as well, as depicted by D, E, and F. (3) The first round purification via specific interaction between the affinity binder 1 and the far-end tag eliminates most nonspecific associated proteins. (4) In the second round purification, the near-end tag is cut off and the protein complex is further purified, with most nonspecific associates removed. (5 and 6) The bait protein and its associated proteins are eluted from the column and are further analyzed either by using antibodies, or by mass spectrometry...

Fig. 14 The tandem affinity purification (TAP) technique, a Schematic representation of the TAP tag. b Overview of the TAP purification strategy. Reproduced from [10] with permission from Birkhauser Verlag, 2006...

Fig. 24 MS-based large-scale analysis of S. cerevisiae s protein-protein interaction network (interactome). Tandem affinity purification (TAP) was used to isolate the interaction partners of 4562 different tagged proteins. Each sample was analyzed twice, by SDS-PAGE-MALDI MS and by LC-ESI MS/MS, to increase coverage and accuracy. In total 7123 protein-protein interactions were identified involving 2708 different proteins and 547 protein complexes, a Summary of the experimental strategy and data analysis. PPI, protein-protein interactions, b-f The proportions of proteins identified as bait or prey are shown in relation to protein mass (b), and known expression level (c) and intracellular localization (d). Reproduced from [399] with permission from Nature Publishing Group, 2006...

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