  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.
