Systematic Quantification of GFP-tagged Protein Foci in Schizosaccharomyces pombe Nuclei

Kim Kiat Lim; Ee Sin Chen

doi:10.21769/BioProtoc.3117

Systematic Quantification of GFP-tagged Protein Foci in Schizosaccharomyces pombe Nuclei

Bio Protoc. 2018 Dec 20;8(24):e3117. doi: 10.21769/BioProtoc.3117.

Authors

Kim Kiat Lim¹, Ee Sin Chen^{1

2

3}

Affiliations

¹ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
² National University Health System, Singapore.
³ NUS Graduate School of Science & Engineering, National University of Singapore, Singapore.

Abstract

DNA damage repair proteins form foci in response to DNA damaging agents. The efficiency and integrity of the DNA repair pathway of a particular eukaryotic (mutant) strain is usually determined by the number of foci formed compared with their wild-type counterpart. Conventionally, focus number is determined visually, and this low accuracy may obscure the identification of a weaker phenotype, particularly when the output is low. Here, using the homologous recombination protein Rhp54 as an example, we present a protocol that can increase the consistency of foci identification among samples and can significantly improve the efficiency of foci quantification for large sample sizes. A similar method can be applied to other foci-forming proteins.

Keywords: DNA damage; Fission yeast; Homologous recombination; Rhp54; Set2.