Extracting, enriching, and identifying nuclear body sub-complexes using label-based quantitative mass spectrometry.
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]
- Published Article
Methods in molecular biology (Clifton, N.J.)
- Publication Date
Jan 01, 2015
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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The corresponding record at NLM can be accessed at https://www.ncbi.nlm.nih.gov/pubmed/25555584