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

Abstract

Measurement of beta-galactosidase (beta-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.

The ROXY1 C-Terminal L**LL Motif Is Essential for the Interaction with TGA Transcription Factors

“…Quantification of b-Galactosidase Activity b-Galactosidase activity from cotransformed or mated yeast was measured using the o-nitrophenyl b-D-galactopyranoside assay, where o-nitrophenyl produced from cleavage of o-nitrophenyl b-D-galactopyranoside by b-galactosidase was detected by spectrophotometry (Miller, 1972;Mö ckli and Auerbach, 2004).…”

Helper Component Proteinase of the Genus Potyvirus Is an Interaction Partner of Translation Initiation Factors eIF(iso)4E and eIF4E and Contains a 4E Binding Motif

“…Experiments were carried out three times. Protein-protein interactions were quantified in the yeast strain Y187 (Clontech) by measurement of ␤-galactosidase activity using the pellet X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) (PXG) assay and processing images with Image J (National Institutes of Health) as described previously (56). The positive control (vector pair supplied by Clontech) was set to 100% ␤-galactosidase activity to determine the relative strength of interactions in other samples.…”

Tyrosine Phosphorylation of the Triple Gene Block Protein 3 Regulates Cell-to-Cell Movement and Protein Interactions of Potato Mop-Top Virus

“…To test the strength of the interactions, yeast strain Y187 was cotransformed with the above-described constructs, and the pellet 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal) ␤-galactosidase (␤-gal) assay was performed and analyzed as described previously (42). The intensity of the color, caused by cleavage of X-gal by ␤-gal activity, was determined with ImageJ software (43).…”

“…Quantitative differences in the strength of interactions were studied by cotransforming the plasmids in the above-mentioned combinations into another yeast strain that encoded ␤-galactosidase under a strong promoter, facilitating ␤-galactosidase mea- surements using a microplate assay (42). Although interactions between TGBp2 and wt TGBp3 or the 21K 120A protein (Fig.…”