Figure 4.
Identification of the GUS-binding region of VASA. (A) (His)6-tag pull-down experiment. Each of the indicated (His)6-tagged MBP-fused VASA deletion mutants and GUS without a tag were mixed and incubated with Ni-NTA agarose resin (QIAGEN). The resin was vigorously washed and subjected to denaturing gel electrophoresis. (B) ITC analysis of the interaction between GUS and VASA. The ITC experiments were carried out by titrating 5 μl of 0.15 mM of GUS into the solution of full-length VASA (5 μM) or titrating 5 μl of 0.2 mM of the 30 amino-acid VASA peptide into the solution of GUS (9 μM) or GUS in complex with ElonginBC (9 μM). KD values were deduced from curve fittings of the integrated heat per mol of added ligand and presented in the table. The titration curves obtained for the formation of the indicated complexes are shown in the insets.