LPS-CD14 Binding Assay Protocol

LPS-CD14 binding assays were performed by incubating 100 ng/mL of recombinant bovine sCD14-His [82 (link)] with various concentrations of leptospiral LPS in PBS for 1 h at 37°C and under slow agitation at 300 rpm. Samples were then loaded on polyacrylamide native gels (stacking 4.5%, running 20%), ran in TG native buffer (BioRad) at constant voltage of 50 V for 6-8 h on ice and used for WB or silver staining. Silver staining was performed using the Pierce coloration kit (ThermoFisher) according to the manufacturer’s instructions. Images were acquired within 30 min of development. WBs were performed after turbo transfer to nitrocellulose membrane (BioRad) and blocking in 5% w/V BSA in PBS-Tween (PBS-T). Membrane was then stained with 1 μg/mL of HRP coupled anti-His (monoclonal mouse anti-polyHistidine-Peroxidase, Sigma-Aldrich) in 1% w/V BSA in PBS-T over night at 4°C. Signal was visualized after three washes and with Clarity ECL reagents (BioRad) using automatic exposure on ChemiDoc imaging system (BioRad).