Media composition influences yeast one- and two-hybrid results
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SHORT REPORT
Biological Procedures Online Open Access
Media composition influences yeast one- and two-hybrid results Ying Liu1†, Zabeena Merchant1†, Hao-Ching Hsiao2†, Kim L Gonzalez2, Kathleen S Matthews1 and Sarah E Bondos1,2*
Abstract Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments. Keywords: Yeast one-hybrid, yeast two-hybrid, protein-protein interaction, accuracy, false positive, false negative
Findings The yeast two-hybrid system provides an efficient method for identifying novel protein·protein interactions in small-scale studies and high-throughput screens [1-3]. In this system, the first hybrid is composed of a bait protein fused to a DNA binding protein that recognizes DNA sequences upstream of a lacZ reporter gene. The second hybrid protein consists of a strong activation domain fused to a potential protein partner. Interaction between bait and partner proteins localizes the strong activation domain to the lacZ promoter, thus activating transcription of the lacZ reporter gene. Multiple cycles of lacZ transcription, translation, and b-galactosidase cleavage of X-gal generate visible quantities of the blue assay product, 5-bromo-4-chloro-3-hydroxyindole. This sensitive system detects interactions that may be difficult to observe by other means (e.g., co-immunoprecipitation) due to low abundance of the protein complex. The sensitivity of this method can also lead to several problems: (i) transformation efficiency affects signal strength and is highly dependent on technique [3], (ii) 25% to 50% of detected interactions are estimated to be
false positives [2,4,5], and (iii) 55% to 90% of true protein interactions are not observed (false negatives) [2,5]. Furthermore, differences in protein abundance can visibly impact outcome [6,7]. Given these difficulties, it is perhaps not surprising that laboratories using similar assays report different protein interactions [2,5]. Identifying sensitive parameters in yeast two-hybrid experiments could help address these difficulties. Herein, we report both the source of media components and the media preparation protocol can impact yeast one-hybrid and two-hybrid results. In the yeast onehybrid system, the bait protein, which includes a transcription activation domain, is fused to a DNA binding protein [1]. When the protein of interest is hybridized to a DNA binding domain, this assay can be used to assess the presence and strength of an activatio
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