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تسجيل مستخدم جديدAnalysis of interaction partners of H4 histone by a new proteomics approach
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We describe a modification of the TAP method for purification and analysis of multiprotein complexes, termed here DEF-TAP (for Differential Elution Fractionation after Tandem Affinity Purification). Its essential new feature is the use for last purification step of 6XHis-Ni++ interaction, which is resistant to a variety of harsh washing conditions, including high ionic strength and presence of organic solvents. This allows us to use various fractionation schemes before the protease digestion, which is expected to improve the coverage of the analysed protein mixture and also to provide an additional insight into the structure of the purified macromolecular complex and the nature of protein-protein interactions involved. We illustrate our new approach by analysis of soluble nuclear complexes containing histone H4 purified from HeLa cells. In particular, we observed different fractionation patterns of HAT1 and RbAp46 proteins as compared to RbAp48 protein, all identified as interaction partners of H4 histone. In addition, we report all components of the licensing MCM2-7 complex and the apoptosis-related DAXX protein among the interaction partners of the soluble H4. Finally, we show that HAT1 requires N-terminal tail of H4 for its stable association with this histone.
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