Use of X-alpha-Gal in the Yeast-two-hybrid System

The yeast two-hybrid screen is a genetic assay to detect protein-protein interactions in vivo. This method is used to investigate the interaction of two known proteins or to identify previously unknown binding partners of specific proteins by screening a library. The method takes advantage of the modular properties of GAL4 and other transcription factors. Many eukaryotic transcription factors have at least two distinct functional domains, one binding to a promoter DNA sequence and another one activating transcription. The DNA-binding domain of GAL4 is not activating transcription unless interacting with the activating domain.

In the experiment, the protein of interest is fused to a DNA-binding domain (the bait) and another protein is fused to the activating domain (the prey). These fusion proteins are co-transfected into an appropriate yeast host cell. If the proteins interact, transcription of the reporter gene will be activated which enables the auxotrophic yeast two-hybrid strains to grow on nutritionally selective media.

Most two-hybrid methods also use the E. coli lacZ gene as a second reporter gene. Usually the colonies growing on the selection plates are assayed for the activation of the reporter gene lacZ in a second screen by a time-consuming filter-lift assay.

Alternatively, in yeast strains producing alpha-galactosidase the chromogenic substrate X-alpha-Gal can be used to assay GAL4-based two-hybrid interactions directly on the nutritional selection plates.
The X-alpha-Gal assay monitors the activation of the yeast MEL1 gene, a GAL4-regulated gene encoding the secreted enzyme alpha-galactosidase which hydrolyzes colorless X-alpha-Gal into a blue end product. This quick and easy assay that takes advantage of the extracellular localization of the alpha-galactosidase eliminates the need for time-consuming beta-galactosidase liquid and filter-lift assays in two-hybrid analysis.


Cat.No. - Product Name
B-7141 - 5-Bromo-4-chloro-3-indoxyl-alpha-D-galactopyranoside, CAS No.[107021-38-5]

Literature:
1. Aho S, Arffman A, Pummi T, Uitto J. A novel reporter gene MEL1 for the yeast two-hybrid system. Anal Biochem. 1997;253(2):270-2.
2. Fields S, Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989;340(6230):245-6.

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