Journal article
A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, Vol.136, e57801
06/01/2018
DOI: 10.3791/57801
PMCID: PMC6101618
PMID: 29939176
Abstract
Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.
Details
- Title: Subtitle
- A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions
- Creators
- Tabitha A. Peterson - University of IowaMark A. Stamnes - University of IowaRobert C. Piper - University of Iowa
- Resource Type
- Journal article
- Publication Details
- JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, Vol.136, e57801
- Publisher
- Journal Of Visualized Experiments
- DOI
- 10.3791/57801
- PMID
- 29939176
- PMCID
- PMC6101618
- ISSN
- 1940-087X
- eISSN
- 1940-087X
- Number of pages
- 17
- Grant note
- R01GM058202 / NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS) 1517110 / NSF; National Science Foundation (NSF) P30CA086862 / NATIONAL CANCER INSTITUTE; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI) 1517110 / Div Of Molecular and Cellular Bioscience; National Science Foundation (NSF); NSF - Directorate for Biological Sciences (BIO) R21EB021870 / NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Biomedical Imaging & Bioengineering (NIBIB) NIH R21 EB021870-01A1 / National Institutes of Health; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA
- Language
- English
- Date published
- 06/01/2018
- Academic Unit
- Molecular Physiology and Biophysics; Medicine Administration; Internal Medicine
- Record Identifier
- 9984297610202771
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