Michael L Goodson, Behnom Farboud, Martin L Privalsky
{"title":"An improved high throughput protein-protein interaction assay for nuclear hormone receptors.","authors":"Michael L Goodson, Behnom Farboud, Martin L Privalsky","doi":"10.1621/nrs.05002","DOIUrl":null,"url":null,"abstract":"<p><p>The Glutathione-S-Transferase (GST) \"pulldown\" assay has been used extensively to assay protein interactions in vitro. This methodology has been especially useful for investigating the interactions of nuclear hormone receptors with a wide variety of their interacting partners and coregulatory proteins. Unfortunately, the original GST-pulldown technique relies on multiple binding, washing and elution steps performed in individual microfuge tubes, and requires repeated centrifugation, aspiration, and suspension steps. This type of batch processing creates a significant liquid handling bottleneck, limiting the number of sample points that can be incorporated into one experiment and producing inherently less efficient washing and elution than would a flow-through methodology. In this manuscript, we describe the adaptation of this GST-pulldown assay to a 96-well filter plate format. The use of a multi-well filter plate makes it possible to assay more samples in significantly less time using less reagents and more efficient sample processing than does the traditional single tube assay.</p>","PeriodicalId":87415,"journal":{"name":"Nuclear receptor signaling","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2007-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1621/nrs.05002","citationCount":"19","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nuclear receptor signaling","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1621/nrs.05002","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 19
Abstract
The Glutathione-S-Transferase (GST) "pulldown" assay has been used extensively to assay protein interactions in vitro. This methodology has been especially useful for investigating the interactions of nuclear hormone receptors with a wide variety of their interacting partners and coregulatory proteins. Unfortunately, the original GST-pulldown technique relies on multiple binding, washing and elution steps performed in individual microfuge tubes, and requires repeated centrifugation, aspiration, and suspension steps. This type of batch processing creates a significant liquid handling bottleneck, limiting the number of sample points that can be incorporated into one experiment and producing inherently less efficient washing and elution than would a flow-through methodology. In this manuscript, we describe the adaptation of this GST-pulldown assay to a 96-well filter plate format. The use of a multi-well filter plate makes it possible to assay more samples in significantly less time using less reagents and more efficient sample processing than does the traditional single tube assay.