Detection of Protein–Protein Interactions Using Tandem Affinity Purification
Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.
Key wordsProtein–protein interactions Tandem affinity purification TAP tagging Affinity purification Affinity tags Proteomics Protein purification
This work was supported by a Wellcome Trust senior fellowship awarded to I.G. and a Research Fellowship from the Pirbright Institute and the University of Birmingham awarded to D.B.
- 6.Cipak L, Spirek M, Novatchkova M, Chen Z, Rumpf C, Lugmayr W, Mechtler K, Ammerer G, Csaszar E, Gregan J (2009) An improved strategy for tandem affinity purification-tagging of Schizosaccharomyces pombe genes. Proteomics 9(20):4825–4828. doi: 10.1002/pmic.200800948 PubMedCentralPubMedCrossRefGoogle Scholar
- 8.Holowaty MN, Zeghouf M, Wu H, Tellam J, Athanasopoulos V, Greenblatt J, Frappier L (2003) Protein profiling with Epstein-Barr nuclear antigen-1 reveals an interaction with the herpesvirus-associated ubiquitin-specific protease HAUSP/USP7. J Biol Chem 278(32):29987–29994. doi: 10.1074/jbc.M303977200 PubMedCrossRefGoogle Scholar
- 9.Rohila JS, Chen M, Chen S, Chen J, Cerny RL, Dardick C, Canlas P, Fujii H, Gribskov M, Kanrar S, Knoflicek L, Stevenson B, Xie M, Xu X, Zheng X, Zhu JK, Ronald P, Fromm ME (2009) Protein–protein interactions of tandem affinity purified protein kinases from rice. PLoS One 4(8):e6685. doi: 10.1371/journal.pone.0006685 PubMedCentralPubMedCrossRefGoogle Scholar
- 11.Li Y, Franklin S, Zhang MJ, Vondriska TM (2011) Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: application to Bruton’s tyrosine kinase. Protein Sci 20(1):140–149. doi: 10.1002/pro.546 PubMedCentralPubMedCrossRefGoogle Scholar
- 12.Guerrero C, Tagwerker C, Kaiser P, Huang L (2006) An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26S proteasome-interacting network. Mol Cell Proteomics 5(2):366–378. doi: 10.1074/mcp.M500303-MCP200 PubMedCrossRefGoogle Scholar