Protocol

Indirect Immunofluorescence Labeling in the Yeast Saccharomyces cerevisiae

Adapted from Basic Methods in Microscopy (eds. Spector and Goldman). CSHL Press, Cold Spring Harbor, NY, USA, 2006.

INTRODUCTION

The budding yeast Saccharomyces cerevisiae is an ideal model system for the initial characterization of novel genes and their corresponding gene products. Genetic analysis is straightforward, and sophisticated cell biological methods are available. Furthermore, only in a genetically tractable organism is it possible to use epitope-tagged proteins to the best advantage. In S. cerevisiae, it is a trivial matter to determine whether a modified version of a protein is functional. This is accomplished by testing whether the tagged protein can complement a null phenotype or rescue a conditional phenotype. Methods such as indirect immunofluorescence and the use of green fluorescent protein (GFP) allow the investigator to correlate the intracellular localization of the protein with its function in vivo. This article includes a detailed protocol for performing indirect immunofluorescence with S. cerevisiae. Cells are grown exponentially and then fixed. After fixation, an enzyme is used to remove the cell wall, and the cells are adhered to slides. Subsequent steps involve the application of primary and secondary antibodies and the final processing of the slide.