We are interested in chromatin structure, so our targets are mostly chromatin-bound proteins, Kireev said.
The researchers had inserted several copies of a bacterial DNA, called the Lac operator, into the chromosomes. A bacterial protein, the Lac repressor, recognizes and binds to the Lac operator in living cells.
The researchers combined a Lac repressor protein with another protein that fluoresces green under blue light. This engineered protein adhered to the chromosomes in regions containing the Lac operator sequences. Under blue light, these regions fluoresced. A gold-tagged antibody targeted against green fluorescent protein (GFP) was then microinjected into the nucleus of a living cell, which added a metallic signal that could be boosted with silver.
All this combined gives us a much better signal, a much stronger signal, with the very best structural preservation, Kireev said.
The fluorescing protein helped the researchers find the regions of interest in the cells. These areas were then immunogold labeled and targeted for electron microscopy.
In the resulting micrographs the researchers saw enhanced staining of the chromosomes.
We can now apply this same live-cell labeling method to study at high resolution many different GFP-tagged proteins in the cell cytoplasm or nucleus, said Andrew Belmont, a professor of cell and developmental biology and senior author of the paper.
In trying to understand chromosomes, people have largely been limited to low resolution visualization of specific chromosomal proteins using light microscopy, Belmost said. This meant everyone has had to
|Contact: Diana Yates|
University of Illinois at Urbana-Champaign