Evaluation of the Nucleolar Localization of the RENT Complex to Ribosomal DNA by Chromatin Immunoprecipitation Assays.
Chromatin immunoprecipitation (ChIP) is a valuable technique for localizing proteins of interest to specific genomic sites and determining the relative abundance of these proteins at these sites. The ChIP method entails chemical cross-linking of proteins to genomic DNA, isolation of protein-DNA conjugates, and purification of DNA from conjugates. Real-time polymerase chain reactions are used to identify and quantify isolated genomic sequences. Here we describe how to localize yeast proteins to gene sequences residing within the nucleolus, i.e., ribosomal DNA (rDNA).
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA. julie_huang@hms.harvard.edu.
Julie
J
Huang
Department of Cell Biology, Howard Hughes Medical Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA. nahid_iglesias@hms.harvard.edu.
Nahid
N
Iglesias
Department of Cell Biology, Howard Hughes Medical Institute, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA.
Danesh
D
Moazed
D018929:Cell Culture Techniques / Q000379:methods; D018797:Cell Cycle Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005557:Formaldehyde / Q000737:chemistry; D016681:Genome, Fungal; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012097:Repressor Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D056565:Sirtuin 2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D002843:Chromatin; D003432:Cross-Linking Reagents; D004275:DNA, Ribosomal; C119300:Net1 protein, S cerevisiae; D009687:Nuclear Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D005557:Formaldehyde; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2
Chromatin immunoprecipitation (ChIP); Net1; Quantitative real-time polymerase chain reaction (qPCR); RENT; Ribosomal DNA (rDNA); Sir2