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Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications
Published on: October 30, 2013
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Budding yeast protein extraction and purification for the study of function, interactions, and post-translational
Eva Paige Szymanski1, Oliver Kerscher
1Integrated Science Center, Biology Department, The College of William & Mary.
Journal of Visualized Experiments : Jove
|December 5, 2013
Summary
Bead mill homogenization efficiently extracts proteins from challenging budding yeast cells, preserving their function and modifications for downstream analysis like Western blotting and purification. This method aids in studying cell cycle proteins and their modifications.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Budding yeast cells are difficult to lyse for molecular analysis.
- Efficient extraction of intact proteins is crucial for studying their functions, interactions, and post-translational modifications.
Purpose of the Study:
- To describe a bead mill homogenization protocol for efficient protein extraction from budding yeast.
- To optimize buffers for maintaining protein integrity and functionality post-extraction.
Main Methods:
- Cells are grown in liquid media, optionally treated with inducers or inhibitors, pelleted, and resuspended in buffer with inhibitors.
- Homogenization is performed using a bead mill (six 20-sec cycles at 5.5 m/sec with 1-min on-ice incubation).
- Extracts are cleared by centrifugation and filtration, then precipitated with TCA for SDS-PAGE and Western blotting, or used for purification.
Main Results:
- The protocol yields cleared whole cell extracts (WCE) suitable for total protein analysis.
- Demonstrated successful extraction and purification of Siz1 (a SUMO E3 ligase) and Slx5 (a ubiquitin ligase subunit).
- The method preserves protein modifications like sumoylation and phosphorylation.
Conclusions:
- Bead mill homogenization is a rapid and effective method for protein extraction from budding yeast.
- The protocol provides a foundation for optimizing protein purification and studying protein modifications.
- This technique is valuable for analyzing cell cycle-regulated proteins and their associated complexes.
