Cycloheximide Assays to Measure Protein Degradation in vivo in Plants

The half-life of a protein is a characteristic property and can be modulated by post-translational modifications, changes in subcellular localization, and/or interaction with other proteins or ligands. As on determinant of its steady-state level, a protein's degradation represents an important distinguishing attribute relevant to its biological function. Because protein longevity cannot be elucidated from bioinformatics analyses, it must be determined empirically. Here we describe two approaches for in vivo half-life determination in plants: 1. pooled-seedling degradation assays monitoring either tagged versions of the protein (luciferase fusions or other epitope tags) or following the endogenous protein; 2. single-seedling degradation assays using luciferase fusion proteins. The advantages of these approaches are their simplicity and low cost.