Lumiglo 1 peroxidase

Fibrinogen (200 ng, Enzymes Research, UK) was separated on 10 % Bis-Tris NuPAGE Gels at 180V for 75 min (ThermoFisher Scientific) with colourmetric protein markers (BioRad Kaleidoscope). The gel was split and a portion was stained with SimplyBlueTM SafeStain (Invitrogen) to determine the positions of the α, β and γ chains. The other portion was transferred in 20 % methanol to PVDF (1 h, 100 V). The membrane was subsequently blocked [5 % (w/v) BSA in PBS-T] overnight at 4 °C, probed with 500 nM rFnBPB or rFnBPB_N277A/F279A in 5 % (w/v) BSA PBS-T for 4 h 20 °C. The membrane was washed three times in PBS, before application of Anti-His₆-Peroxidase (Roche, 1 : 1000) in 5 % (v/w) BSA PBS-T. The membrane was washed three times in PBS, before 1 min incubation in 1×LumiGLO/1×Peroxidase (CellSignal). Blots were visualised in an Amersham A680 imager.

SH1000 clfAclfBfnbAfnbB strains carrying variants of the pALC2073 vector were grown overnight in TSB supplemented with chloramphenicol. The cells were washed twice in TSB before being diluted 1 : 100 in pre-warmed TSB supplemented with chloramphenicol and grown at 37 °C 200 r.p.m. Cells were induced at OD₆₀₀=0.18 with 300 ng ml⁻¹ ATc. Cells were grown until OD₆₀₀=0.35 when they were harvested and washed in equal volume of PBS. The OD₆₀₀ was adjusted to 10. 1 ml fraction was pelleted and resuspended in EDTA-free protease inhibitor cocktail (Roche), lysostaphin (12.5 µg), 50 mM Tris-HCl (pH 7.5), 20 mM magnesium chloride and incubated for 8 min at 37 °C. The cell debris was pelleted. The supernatent was boiled with Laemmli sample buffer (Sigma) before loading on to 10 % Bis-Tris SDS-PAGE gels. Proteins were transferred to PVDF and blocked with 10 % Skimmed Milk Powder in Tris-buffered saline (TBS). The membrane was subsequently probed with rabbit serum specific for the FnBPB A-domain residues 163–480 [10 (link)], 1 : 1000 in 10 % Skimmed Milk in TBS. The membrane was washed three times in TBS before being probed with protein A-peroxidase (Merck, 1 : 50 000 in 10 % Skimmed Milk in TBS). The membrane was incubated with 1×LumiGLO/1×Peroxidase (CellSignal) for 1 min before imaging in a GE Amersham imager AI680.