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The DNA damage-dependent checkpoint of Saccharomyces cerevisiae is a paradigm for eukaryotic checkpoint pathways that regulate cell cycle progression in the presence of insults to the genetic material. In order to better understand this pathway, we undertook a biochemical study of the proteins implicated in its functioning. Analysis of the hydrodynamic properties of a protein in a crude mixture can give insights into possible tertiary organization such as participation in high-molecular-mass protein complexes. We here describe the determination of Stokes radius and sedimentation coefficients for the Rad24 protein, which enabled us to predict that this protein was a component of a protein complex in crude yeast extracts. This led us to develop a protocol to purify this complex to homogeneity in order to determine the component proteins. The methods described here should be applicable to the hydrodynamic analysis and subsequent purification of any soluble protein from organisms amenable to genetic manipulation, such as yeast, as long as the function of that protein is not perturbed by the addition of an epitope tag.

Original publication

DOI

10.1385/1-59259-788-2:291

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2004

Volume

280

Pages

291 - 306

Keywords

Blotting, Western, Cell Cycle, Cell Cycle Proteins, Cell Division, Chromatography, Gel, DNA Damage, Dose-Response Relationship, Radiation, Epitopes, Heparin, Intracellular Signaling Peptides and Proteins, Nickel, Protein Structure, Tertiary, Saccharomyces cerevisiae, Sepharose