Extracting, Enriching, and Identifying Nuclear Body Sub-Complexes Using Label-Based Quantitative Mass Spectrometry.
Nuclear Bodies and Noncoding RNAs
Methods in molecular biology, January 2015
Fox A, Mehta V, Boulon S, Trinkle-Mulcahy L, Archa Fox, Virja Mehta, Severine Boulon, Laura Trinkle-Mulcahy
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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