SEM image of a mammalian nucleus, with arrow pointing to nucleolus

SEM image of purified nucleoli

Due to its inherent high density, nucleoli from cultured human cells can be isolated in great quantities from sonicated nuclear extracts. Taking advantage of this, our laboratory and others have previously employed various mass spectrometry techniques to identify the protein components from highly purified nucleolar preparations. Visit the Protocols page on the Lamond Lab website for a detailed nucleolar isolation protocol, including still images and Quicktime movies demonstrating key steps.

Fluorescent protein-tagging experiments and photobleaching analyses have demonstrated the dynamic nature of the nucleolar proteome, where proteins accumulate in the nucleolus only under specific metabolic conditions or at specific cell cycle stages.

Recently, we have extended our mass spectrometry analyses to measure the dynamic behaviour of the nucleolar proteome by quantitating the relative level of individual nucleolar components upon transcriptional inhibition using a method known as Stable Isotope Labelling with Amino acids in Cell Culture (SILAC).

DIC image of a nucleolus following inhibition of transcription (green is overlay of GFP-PSP1, which accumulates in "caps" at the periphery of the nucleolus)