Anti his hrp

ELISAs were carried out in clear, flat-bottomed 96-well MaxiSorp plates obtained from Thermo-Fisher Scientific,UK. SARS-CoV-2 protein (either spike or RBD) was prepared at 2 µg per mL in PBS and 100 µL per well added to an ELISA plate alongside negative control wells with 100 µL PBS alone. Plates were then incubated for 16 h at 4 C and washed three times with 0.05% v/v Tween-20 in PBS (PBS-T) prior to addition of 150 µL of 5% w/v BSA dissolved in PBS and room temperature (RT) incubation for 4 h. Blocked wells were then washed 2 times with PBS-T, followed by the addition of 100 µL of His-tagged antibody diluted in PBS-T and the plate incubated for 16 h at 4 C. Wells were then washed 4 times with PBS-T and incubated with 100 µL of anti-His-HRP (mouse monoclonal anti-polyhistidine antibody (clone HIS-1) conjugated to peroxidase (anti-His-HRP) was obtained from Sigma-Aldrich) diluted 1:4000 with 1% w/v BSA in PBS for 2 h at RT, followed by washing 4 times with PBS-T. 25 µL of TMB was added to each well, incubated for 20 min at RT, then 25 µL 3 M HCl added and absorbance read at 450λ.

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).