His magnetic beads

In vitro protein complex formation was assayed using His-Magnetic beads (Promega). All reactions were conducted in interaction buffer containing 50 mm Tris-HCl, pH 8, 100 mm NaCl, 5 mm MgCl₂, 0.05% Tween 20 (v/v), 10% glycerol (v/v), 20 mm imidazole in the presence or absence of effectors (ATP, ADP, and/or 2-OG) as indicated in each experiment. Five microliters of beads were equilibrated in 200 μl of interaction buffer. The beads were recovered and resuspended in 400 μl of buffer followed by the addition of 20 μg of His-PII and 40 μg of untagged NadE protein, in this order. After 5 min at room temperature with gentle mixing, the beads were washed three times with 200 μl of interaction buffer. Bound proteins were eluted by boiling the beads in SDS-PAGE sample buffer. Proteins were analyzed by Coomassie Blue–stained 15% SDS-PAGE.

The in vitro interaction between CyaC, Clr and CycR was tested via co-elution of respective protein pairs using HisMagnetic beads (Promega) as described before [22 (link)]. In each experimental set-up one of the proteins was provided with a His₆-tag. HisMagnetic beads (5 µl) were equilibrated with 200 µl buffer 1 (50 mM K-phosphate, 150 mM NaCl, in the presence or absence of 0.5 mM cAMP and 3.5 mM ATP as indicated, at pH 7.5). The His₆-and the Strep-tagged proteins (2 µM each in 500 µl buffer 1) were incubated at 30 °C and 1,000 rpm for 10 min, mixed with equilibrated HisMagnetic beads (5 µl beads in 200 ml buffer 1) and incubated for further 5 min at 30 °C under gentle movement (1,000 rpm). The beads were washed twice with 300 µl buffer 1, and the proteins were eluted with 20 µl buffer 1 containing imidazole (0.5 M) for 5 min. Eluted samples were mixed with SDS-PAGE sample buffer and analyzed via SDS-PAGE using 15% gels and finally detected by Western blotting using the respective antibodies [21 (link), 23 (link)]. Immunostaining was performed with horse-radish peroxidase (HRP)-coupled anti-His-HRP, anti-Strep-HRP, and anti-IgG-mouse-HRP polyclonal antiserum (Sigma-Aldrich) or with anti-PhoA antibodies (produced in mouse, Sigma-Aldrich). For visualization the blots were subjected to a chemiluminescent substrate (HRP; Merck Millipore) and exposed on X-ray films (Advansta).