Authors
Talat Nasim and Richard Trembath
Summary
Methods to investigate protein-protein interactions in mammalian cells are limited by the use of single reporter functions1-5. Such assays are susceptible to numerous intrinsic variables in the level of transfection efficiency, transcription and translation. We have developed assay systems based on enzymatic and fluorescence activities that determine the efficiency of protein-protein interactions in mammalian cells and overcome the potential confounders of single reporter methods6. The approach utilizes two gene expression units linked to reporter functions interposed by a deactivation-activation unit such that the downstream unit is switched off. In the event of an interaction the downstream unit is switched on leading to two reporter functions, whilst the upstream reporter is expressed regardless of protein-protein interactions. Thus, the ratio of two reporter activities provides a reference standard to determine the efficiency of protein-protein interactions. The enzymatic assay provides a quantitative analysis, whilst the dual-fluorescence assay is rapid. The dual-fluorescence assay is directly measured on intact cell and does not require cell wall disruption or addition of a substrate and the sensitivity is comparable to that with gal-luc assay and therefore is suitable for high-throughput screening of cDNA, small molecules and peptides that might affect a specific protein-protein interaction.Further details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.Advertisement
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