(A)
In vitro, protein-protein interactions indicate that SinR’-MD binds tightly to SinR. E. coli whole cell lysates expressing various GST-tagged SinR’ proteins were incubated with SinR-6His proteins and were purified using Ni++ agarose affinity columns. The elutes were probed with Rabbit Anti-GST and with Mouse Anti-His antibodies. Lanes details are as follows:
I. Input 1: Mixture of SinR’-GST expressing E. coli lysate with purified SinR-6His
UB. Unbound from input 1 after passing through Ni++ column
W. First wash
E1. Elute with 50 mM imidazole (GST+SinR-6His)
E2. Elute with 200 mM imidazole (SinR’-GST + SinR-6His)