2.9 | Co-elution of EspB and
EspD35-His by nickel affinity chromatography
Co-elution assays were performed as previously described (Luo &
Donnenberg, 2011). Briefly, EPEC ΔespD in the presence or the
absence of an EspD-35His expression vector, was grown
under T3SS-inducing conditions for 7 hr (0.5 mM IPTG was added after 3
hr to induce protein expression). To evaluate the ability of mAb-EspB-B7
to inhibit the interaction between EspB and EspD, 100 or 200 nM of
mAb-EspB-B7 were added to EPEC ΔespD expressing
EspD-35His sample. The supernatants, containing
secreted EspD-35His and EspB, were collected by
centrifugation (20000 × g for 5 min) and were passed through a
0.45-μm-pore-size filter. Protease inhibitor solution was added to the
samples (200 mM PMSF and 1 µM benzamidine), and they were incubated with
Ni-NTA resin while being rotated overnight at 4°C. The samples were then
loaded on gravity columns, and the flow-through was collected. The
columns were washed three times with 5 mL of washing buffer (30 mM
phosphate buffer pH 7.5, 500 mM NaCl, 50 mM imidazole), and proteins
were eluted using elution buffer (30 mM phosphate buffer pH 7.5, 500 mM
NaCl, 500 mM imidazole). Equal volumes of the supernatant and the eluate
samples were precipitated with 10% (v/v) TCA for 1 hr at 4°C,
centrifuged (30 min, 16000 × g , 4°C), air dried, and dissolved in
SDS-PAGE sample buffer. Supernatants and eluted samples were analyzed by
SDS-PAGE and western blotting using mouse anti-His and mouse anti-EspB
antibodies, to avoid detection of the human mAB-EspB-B7 antibody.