Abstract
14-3-3s are a highly conserved protein family that exert many regulatory roles in eukaryotic cells by binding to phosphopeptide motifs in diverse target proteins. Here, we describe 14-3-3 affinity binding procedures that can be used to purify and identify 14-3-3-binding phosphoproteins; monitor how their phosphorylation and 14-3-3 binding is regulated by extracellular stimuli; define the functional effects of 14-3-3s on individual targets; and identify relevant protein phosphatases and kinases. In principle, these methods could be adapted to characterize other types of protein-protein interaction that depend on covalent modification of target sites.
Original language | English |
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Pages (from-to) | 469-478 |
Number of pages | 10 |
Journal | Methods in Molecular Biology (Clifton, N.J.) |
Volume | 261 |
DOIs | |
Publication status | Published - 1 Jan 2004 |
Keywords
- Protein-protein interactions
- affinity chromatography
- phosphorylation
- phosphopeptide
ASJC Scopus subject areas
- Molecular Biology
- Genetics