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The two main methods for finding protein–protein interactions are the yeast two-hybrid (Y2H) system and co-immunoprecipitation followed by mass spectrometry. Several companies sell reagents for both approaches. Invitrogen of Carlsbad, California, sells the ProQuest Two-Hybrid System with Gateway Technology. This is based on Y2H, with modifications to decrease false-positive results and allow rapid characterization, says the company. Other firms provide vectors used to produce proteins with affinity tags, which can easily be immunoprecipitated along with other interacting proteins. A polypeptide tag called Flag is popular among researchers, and Sigma Aldrich of St Louis, Missouri, provides several Flag-genes for purchase. Promega in Madison, Wisconsin, has the HaloTag technology, in which a protein of interest is expressed in fusion with a tag protein engineered from a bacterial enzyme. This tag can be used to purify the protein, and any interacting with it, by binding to a resin. The tag is cleaved off using a protease.

Methods such as the yeast two-hybrid system allow scientists to work out which proteins interact. Credit: HYBRIGENICS

For researchers who don't have the time or infrastructure to do the experiments, companies such as Hybrigenics in Paris and Dualsystems Biotech of Schlieren, Switzerland, offer Y2H-based screening. “We have complex libraries with ten times more independent clones than most other libraries, which we screen to saturation. And rather than screening full-length proteins, we screen for interactions with domains,” says Etienne Formstecher, director of scientific projects and sales at Hybrigenics. “Full-length proteins can have some domains buried and not available to interact, at least in yeast where you may not have signals to unlock a closed protein conformation.” A customer is given a list of proteins that interact with the protein of interest; it indicates which domains are making contact and provides a confidence score for each interaction.

Innoprot in Derio, Spain, provides an interaction service using tag-based purification designed for high-throughput analysis. And Invitrogen's ProtoArray Protein–Protein Interaction Service uses microarrays containing more than 9,000 human proteins to identify proteins that interact with any protein of interest.

L.B.