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BacMam-Enabled Cellular Assays Measuring Histone Posttranslational Modifications (Presentation)Télécharger
May 05, 2009
Authors: Roland Leathers, Kevin Lowitz, Coby Carlson, Thomas Machleidt, Kun Bi, Justin Wetter and Matthew Robers, Invitrogen, Madison, Wisconsin; Peter Banks, BioTek Instruments, Winooski, VT
The eukaryotic nucleosome, composed of histones H2A, H2B, H3, and H4, regulates the structure of chromatin and consequently modulates gene transcription profiles in a concerted manner. Nucleosome function is directly regulated by a multitude of posttranslational modifications on amino-terminal tails of core histones, including acetylation, phosphorylation, methylation and ubiquitination. These modifications occur in a tightly regulated fashion, and affect numerous cellular processes including histone deposition, chromatin assembly, and chromosome condensation during both mitosis and meiosis. Therefore, a number of histone modifying enzymes have been identified as valuable targets for therapeutic intervention. The combination of Baculovirusmediated gene delivery (BacMam) with LanthaScreen® cellular assay technology and measurements using the BioTek Synergy™ 4 Hybrid Multi-Mode Microplate Reader, enables a powerful platform for the analysis of target-specific posttranslational modifications of histones in the cell line of interest. Specifically, we have developed HTS-compatible cellular assays measuring acetylation of histone H3 at Lys9, phosphorylation of histone H3 at Ser10, and the ubiquitination status of histone H2B. These assays together with sensitive filter-based detection on the Synergy™ 4 represent a flexible method to measure the ability of compounds to affect histone posttranslational modifications in a HTS-compatible format.