Detection of protein-protein interactions using tandem affinity purification

Research output: Contribution to journalArticlepeer-review

Authors

Colleges, School and Institutes

Abstract

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.

Details

Original languageEnglish
Pages (from-to)121-33
Number of pages13
JournalMethods in molecular biology
Volume1177
Publication statusPublished - 2014

Keywords

  • Carrier Proteins, Chromatography, Affinity, Endopeptidases, Molecular Biology, Protein Binding, Protein Interaction Maps, Proteins, Proteomics