Abstract
An in situ technique for studying the chromatin binding of proteins in single fission yeast cells (Schizosaccharomyces pombe) is described. Cells are permeabilized by enzymatic digestion and extracted with a detergent-containing buffer. This procedure removes soluble proteins, but proteins that are bound to insoluble cell structures such as chromatin are retained, and overall cell morphology is maintained. Extraction of proteins is monitored by fluorescence microscopy, either using fluorescently tagged proteins or by indirect immunofluorescence. This method allows the chromatin association of proteins to be correlated with other cell cycle events without the need for cell synchronization.
MeSH terms
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Amino Acid Sequence
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Base Sequence
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Chromatin / genetics
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Chromatin / metabolism*
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DNA, Fungal / genetics
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DNA, Recombinant / genetics
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Detergents
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Flow Cytometry
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Genetic Vectors
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Micrococcal Nuclease
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Molecular Biology / methods
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Molecular Sequence Data
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Protein Binding
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Schizosaccharomyces / genetics
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Schizosaccharomyces / metabolism*
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Schizosaccharomyces pombe Proteins / genetics
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Schizosaccharomyces pombe Proteins / metabolism*
Substances
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Chromatin
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DNA, Fungal
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DNA, Recombinant
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Detergents
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Recombinant Fusion Proteins
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Schizosaccharomyces pombe Proteins
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Green Fluorescent Proteins
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Micrococcal Nuclease