Efficient extraction of nucleolar proteins for interactome analyses.
The efficient extraction of proteins from purified cellular organelles is critical for in vitro analyses, including identification of protein complex members by affinity purification-based quantitative proteomic approaches. When applied to purified nucleoli, classic nuclear protein extraction methods inefficiently and selectively release only approximately 50% of proteins. Here, we present a method that can extract up to 90% of nucleolar proteins, and apply it in a quantitative interactomic approach to identify nucleolar interaction partners for a mammalian protein phosphatase.
Chamousset
Delphine
D
Department of Cellular & Molecular Medicine, University of Ottawa, Ottawa, ON, Canada.
Mamane
Sarah
S
Boisvert
François-Michel
FM
Trinkle-Mulcahy
Laura
L
D002247:Carbon Isotopes; D002466:Cell Nucleolus / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D009687:Nuclear Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D014357:Trypsin / Q000378:metabolism
D002247:Carbon Isotopes; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010749:Phosphoprotein Phosphatases; D014357:Trypsin