Surface Spreading and Immunostaining of Yeast Chromosomes.
The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.
Department of Radiation and Cellular Oncology, University of Chicago.
Jennifer
J
Grubb
Department of Radiation and Cellular Oncology, University of Chicago; Department of Molecular Genetics and Cell Biology, University of Chicago.
M Scott
MS
Brown
Department of Radiation and Cellular Oncology, University of Chicago; Department of Molecular Genetics and Cell Biology, University of Chicago; dbishop@uchicago.edu.
Douglas K
DK
Bishop
D015825:Chromosomes, Fungal / Q000737:chemistry*; D007158:Immunologic Techniques / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure*