Authors
Masahiro Okada and Tatsuo Fukagawa
Summary
Centromeres contain a multi-protein structure termed the kinetochore that is responsible for faithful chromosome segregation1-5. To understand centromere architecture in vertebrate cells, it is necessary to identify all centromere components in vertebrate cells. Purification of kinetochore protein complexes is difficult due to the low amounts of these proteins and the biochemical characteristics of these proteins. There are relatively few proteins at the inner kinetochore plate, and kinetochore proteins associate tightly with the chromatin. Therefore, it is important to solubilize efficiently protein complexes for immunoprecipitation. We developed a procedure for isolation of the chromatin bound proteins, by which we identified a kinetochore protein complex (CENP-H/I complex) that contains 11 components6. Others have also reported purification of centromere protein complexes and identification of several proteins from such complexes7-11. These researchers used techniques similar to ours; however, specific conditions for protein purification, including the method for solubilization of protein complexes, differ. The optimal condition for each protein complex must be determined. Although our protocol was used to isolate protein complexes that associate with the kinetochores in chicken DT40 cells6, our method should be widely applicable for isolation of protein complexes that associate with chromatin from other cell lines, including human cells.Further details
The protocol was published on Protocol Exchange in 2006. To see the entire protocol, click on the source link.Advertisement
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