Optimized Detection of Protein-Protein and Protein-DNA Interactions, with Particular Application to Plant Telomeres
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
- Keywords
- Bimolecular fluorescence complementation, ChIP for low-abundance proteins, Co-immunoprecipitation, Yeast two-hybrid system,
- MeSH
- Arabidopsis genetics metabolism MeSH
- Cell Nucleus metabolism MeSH
- Chromatin metabolism MeSH
- Chromatin Immunoprecipitation methods MeSH
- DNA, Plant metabolism MeSH
- DNA-Binding Proteins isolation & purification metabolism MeSH
- Immunoprecipitation methods MeSH
- Protein Interaction Mapping methods MeSH
- Optical Imaging methods MeSH
- Arabidopsis Proteins genetics metabolism MeSH
- Two-Hybrid System Techniques MeSH
- Telomerase metabolism MeSH
- Telomere metabolism MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Chromatin MeSH
- DNA, Plant MeSH
- DNA-Binding Proteins MeSH
- Arabidopsis Proteins MeSH
- Telomerase MeSH
Characterization of protein-protein and protein-DNA interactions is critical to understand mechanisms governing the biology of cells. Here we describe optimized methods and their mutual combinations for this purpose: bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), yeast two-hybrid systems (Y2H), and chromatin immunoprecipitation (ChIP). These improved protocols detect trimeric complexes in which two proteins of interest interact indirectly via a protein sandwiched between them. They also allow isolation of low-abundance chromatin proteins and confirmation that proteins of interest are associated with specific DNA sequences, for example telomeric tracts. Here we describe these methods and their application to map interactions of several telomere- and telomerase-associated proteins and to purify a sufficient amount of chromatin from Arabidopsis thaliana for further investigations (e.g., next-generation sequencing, hybridization).
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