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Extracting, enriching, and identifying nuclear body sub-complexes using label-based quantitative mass spectrometry.

Authors
  • 1
  • 1 Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands and Centre for Medical Research, the University of Western Australia, Crawley, Western Australia, 6009, Australia, [email protected] , (Australia)
Type
Published Article
Journal
Methods in molecular biology (Clifton, N.J.)
1940-6029
Publication Date
Volume
1262
Pages
215–238
Identifiers
DOI: 10.1007/978-1-4939-2253-6_13
PMID: 25555584
Source
Medline
License
Unknown

Abstract

Determining the proteome of a nuclear body is a crucial step toward understanding its function; however, it is extremely challenging to obtain pure nuclear body preparations. Moreover, many nuclear proteins dynamically associate with multiple bodies and subnuclear compartments, confounding analysis. We have found that a more practical approach is to carry out affinity purification of nuclear body sub-complexes via the use of tagged nuclear-body-specific marker proteins. Here we describe in detail the method to identify new nuclear body protein sub-complexes through SILAC (stable isotope labeling by amino acids in culture)-based affinity purification followed by quantitative mass spectrometry.

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