Localizing MEN Components by Indirect Immunofluorescence Analysis of Budding Yeast.
The budding yeast Saccharomyces cerevisiae is a very powerful genetic model that has been extensively used in cell cycle studies. Despite the fact that its small size has made imaging studies challenging (haploid cells have a diameter of approximately 4-5 μm that is very close to the maximal optical microscope resolution, ca. 0.20-0.25 μm), the continual improvement of imaging tags and techniques has made it possible to visualize organelles and macromolecules also in this organism. The possibility to easily epitope-tag endogenous proteins and follow them during synchronized cell cycles has proved critical for understanding the distribution of Mitotic Exit Network (MEN) components and gathering insights into their regulation. In this chapter, we describe a detailed protocol for indirect immunofluorescence of fixed cells outlining fixation strategies, cell wall digestion, and the use of primary and secondary antibodies conjugated to fluorescent moieties. This protocol can be used to successfully localize endogenously expressed yeast proteins including MEN components.
Department of Experimental Oncology, European Institute of Oncology, via Adamello 16, Milan, 20139, Italy.
Francesca
F
Senic-Matuglia
Department of Experimental Oncology, European Institute of Oncology, via Adamello 16, Milan, 20139, Italy. rosella.visintin@ieo.eu.
Rosella
R
Visintin
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000032:analysis; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008938:Mitosis; D017346:Protein Serine-Threonine Kinases / Q000032:analysis; D017027:Protein Tyrosine Phosphatases / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis; D014404:Tubulin / Q000032:analysis
C074860:CDC14 protein, S cerevisiae; D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; C085768:DBF2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D017027:Protein Tyrosine Phosphatases
Fluorescent microscopy; Indirect immunofluorescence; MEN; Yeast