Tandem affinity purification

Graumann, J., Dunipace, L.A., Seol, J.H., McDonald, W.H., Yates, J.R., 3rd Wold, B.J., Deshaies, RJ. (2004). Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast. Mol. Cell. Proteomics 3, 226-237. 

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. 

Expression of a recombinant protein using an inducible vector system would permit expression at endogenous levels to simulate physiologic levels of expression of a protein of interest. Tandem affinity purification strategies have recently been employed and facilitate the analyses of highly interactive proteins when the bait protein is expressed at endogenous levels. Immunoaffinity or immunoprecipitation followed by LC-MS/MS does not readily permit determination of the stoichiometry of interacting partners. Additionally, when compared to yeast hybrid experiments, it is difficult to determine whether interactions are binary when identified in complexes by MS/MS. 

Shevchenko, A., Schaet, D., Roguev, A., PijNAPPEL, W. W., Stewart, A. F., Shevchenko, A. (2002). Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry analytical perspective. Mol. Cell Proteomics 1, 204-212. 

Tandem affinity purification also transporter associated with antigen processing... 

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. 

Cheeseman, I.M. and Desai, A. (2005) A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci. STKE 2005, 11. 

Rubio, V., Shen, Y., Saijo, Y., Liu, Y., Gusmaroli, G., Dinesh-Ktrmar, S.P. and Deng, X.W. (2005) An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation. Plant J. 4J, 767-778. 

A slightly modified approach was applied in the identification of protein complexes in Schizosaccharomyces pombe [120]. The major modification involves the use of a tandem affinity purification [121]. To a protein of interest, an epitope is appended that contains two different affinity tags separated by a specific protease cleavage site. This enables isolation of the complex under milder conditions. 

K.L. Gould, L. Ren, A,S. Feoktistova, J.L. Jennings, A.J. Link, Tandem affinity purification and identification of protein complex components. Methods, 33 (2004) 239. 

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. 

Using proteomic technologies (tandem affinity purification and MS) to discover protein-protein interactions, a substantial number of proteins have been identified as potential SPT2 (LCB2)-associated proteins in Saccharomyces cerevisiae (A.C. Gavin,... 

Deciphering protein networks by tandem affinity purification... 

Li Q, Dai X-Q, Shen PY, Cantiello HP, Karpinski E, et al. (2004) A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel. FEBS Letters 576 231-236. 

Characterization of Piant Poiyadenyiation Compiexes by Using Tandem Affinity Purification... 

Key words Pre-mRNA, Poiyadenyiation fector. Tandem affinity purification, Calmodulin binding... 

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...

Rohila JS, Chen M, Cerny R, Fromm ME (2004) Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. Plant J 38(1) 172-181.doi 10.1111/j.l365-313X.2004.02031.x... 

Tandem affinity purification-tag coupied to mass spectrometry (TAP-MS)... 

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