Quantitative β-galactosidase assay suitable for high-throughput applications in the yeast two-hybrid system

Abstract

Measurement of β-galactosidase (β-gal) activity is an important step in every yeast two-hybrid assay, yet many commonly used methods have distinct disadvantages, such as being only qualitative, time-consuming, and cumbersome when processing large numbers of samples. To overcome these drawbacks, we have implemented a novel technique, termed pellet X-gal assay, that allows simultaneous quantitative measurements from large numbers of samples with a minimum of hands-on time. The method was tested using five different, previously described protein-protein interactions and compared to two standard methods, the colony filter lift and the liquid ONPG assay. Our assay allows accurate quantitative measurements of protein-protein interactions and covers a greater dynamic range than the classic ONPG assay. The novel assay is robust and requires very little handling, making it suitable for applications in which several hundreds of individual protein interaction pairs need to be measured simultaneously.