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Methods Mol Biol. 2015;1262:215-38. doi: 10.1007/978-1-4939-2253-6_13.

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

Author information

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, archa.fox@perkins.uwa.edu.au.

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.

PMID:
25555584
DOI:
10.1007/978-1-4939-2253-6_13
[Indexed for MEDLINE]

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