FIG. 4.

Dot blot overlay analysis of the interactions between HasD and HasA(1-174) mutant proteins. His-tagged HasD was overexpressed and purified by affinity chromatography as described in Materials and Methods. HasA(1-174) and derivative mutant proteins were purified by anion-exchange chromatography, followed by gel filtration. Dots of (His)HasD were incubated with HasA(1-174) or a derivative mutant protein for 1 h. After extensive washing, the HasD-HasA(1-174) complexes were detected with a rabbit anti-HasA polyclonal antiserum used at a dilution of 1:5,000. Note that the data generated are qualitative and not quantitative. Each assay was repeated three times.