FIG. 2.
Autophosphorylation of the kinase domain MBP-HrrSΔ1-248 and phosphoryl group transfer to the response regulator HrrA. Purified MBP-HrrSΔ1-248 (12 μM final concentration) was incubated with [γ-33P]ATP for 20 min (A). Then the purified response regulators HrrA was added (12 μM final concentration), resulting in a 2-fold dilution of MBP-HrrSΔ1-248 and the ATP. The samples were incubated at room temperature for a further 60 min. At the indicated time points, samples were taken, mixed with SDS loading buffer, and stored on ice (see Materials and Methods). Finally, these samples were separated by SDS-PAGE and the dried gels were analyzed with a phosphorimager.