Extracting, Enriching, and Identifying Nuclear Body Sub-Complexes Using Label-Based Quantitative Mass Spectrometry

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.

Source: Springer protocols feed by Genetics/Genomics - January 1, 2015 Category: Genetics & Stem Cells Source Type: news