Method to screen for relevant yeast two-hybrid-derived clones by coimmunoprecipitation and colocalization of epitope-tagged fragments--application to Bcl-xL.
The yeast two-hybrid system is a powerful genetic method to screen cDNA libraries to identify protein-protein interactions. A screen with a typical bait may yield many clones, including ones that are not biologically relevant which need to be eliminated by evaluating each clone in an alternative assay to confirm the interaction with the bait protein. We have developed an efficient assay to quickly screen two-hybrid-derived clones for coimmunoprecipitation and colocalization with the bait protein in mammalian cells. Gene fragments derived from a two-hybrid screen are cloned into an epitope tagging vector that can generate high levels of epitope-tagged protein in mammalian cells. The vector expressing an epitope-tagged protein is then cotransfected into mammalian cells with an expression vector for the bait protein. Interaction between the bait protein and epitope-tagged protein is evaluated by coimmunoprecipitation and colocalization. We demonstrate the utility of this approach by applying it to clones isolated in a two-hybrid screen using Bcl-xL as bait, showing that two-hybrid-derived fragments of Bad and Bax, previously known to interact with Bcl-xL, both colocalize and coimmunoprecipitate with Bcl-xL.
Wong
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Department of Pediatrics, Stanford Medical Center, California 94305, USA.
Naumovski
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D000818:Animals; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism; D002999:Clone Cells; D003001:Cloning, Molecular / Q000379:methods*; D000939:Epitopes / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000276:immunology; D006801:Humans; D006822:Hybrid Cells / Q000502:physiology*; D011233:Precipitin Tests / Q000379:methods; D011506:Proteins / Q000276:immunology / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000276:immunology / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015003:Yeasts / Q000235:genetics*; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
C494819:BAD protein, human; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D000939:Epitopes; D011506:Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein