Methods For Visualizing Protein Dynamics Described

The first protocol describes how to insert multiple copies of the bacterial lac operator sequence into the chromosomes of mammalian cells. The protocol includes strategies for stabilizing the repeats and for isolating specific cell clones that are tagged at the chromosomal sites of interest.

Once the tagged cell lines have been established, the chromosomal regions of interest can be visualized with a fluorescently labeled Lac repressor protein, which binds to the lac operator sequence. This allows the scientists to examine the structure of the chromatin and to observe the activity of the chromosomes during replication and transcription.

The protocol was developed by Dr. Andrew Belmont's group from the University of Illinois at Urbana-Champaign . Belmont's lab and others have used this technique to investigate the structure and dynamics of chromatin in live cell cultures, yeast, and various multicellular organisms, including C. elegans, Drosophila, and Arabidopsis.

The second featured method describes how to perform immunohistochemistry in whole mouse embryos, which allows scientists to examine the three-dimensional distribution of a protein during specific stages of development. It can be used to detect endogenous proteins as well as the products of transgenes.

The immunohistochemistry protocol was derived from methods used to examine embryonic development in other species, including Xenopus and Drosophila. It describes several tricks to chemically and physically manipulate the embryos so that the antibodies--which detect the protein of interest--can efficiently penetrate the embryonic tissue.

These methods are freely accessible online (http://www.cshprotocols.org).