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Published on: May 5, 2020
Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation
Y John Shyu1, Susan M Hiatt, Holli M Duren
1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-2091, USA.
Nature Protocols
|April 5, 2008
Summary
This study presents a Venus-based bimolecular fluorescence complementation (BiFC) assay protocol for visualizing protein interactions in living Caenorhabditis elegans. The method allows for rapid detection of protein interactions within 30 minutes of induction in transgenic worms.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Protein interactions are crucial for cellular functions.
- Visualizing protein interactions in vivo is essential for understanding biological processes.
- The bimolecular fluorescence complementation (BiFC) assay offers a method for detecting protein-protein interactions in living cells.
Purpose of the Study:
- To provide a detailed protocol for a Venus-based BiFC assay in the nematode Caenorhabditis elegans.
- To enable visualization of protein interactions in living C. elegans.
- To establish a method for designing BiFC vectors and analyzing results in transgenic worms.
Main Methods:
- Designing C. elegans BiFC cloning vectors with inducible or native promoters.
- Transforming constructs into C. elegans via microinjection.
- Inducing BiFC fusion protein expression using heat shock.
- Analyzing and interpreting fluorescent signals in transgenic animals.
Main Results:
- Successful implementation of a Venus-based BiFC assay in C. elegans.
- Fluorescent signals indicating protein interactions visualized as early as 30 minutes post-induction.
- Fluorescence signals persisted for up to 24 hours in transgenic animals.
- The entire protocol can be completed within 2-3 weeks.
Conclusions:
- The Venus-based BiFC assay is an effective tool for visualizing protein interactions in living C. elegans.
- This protocol facilitates the study of dynamic protein interactions in a whole-organism context.
- The assay's speed and duration allow for detailed temporal analysis of protein interactions.
