In vitro bacterial binding assay

Proteins CLEC-4-His (50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0), CLEC-41-His, and CLEC-42-His (both in 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, 0.5 M L-arginine, pH 8.0) were commercially obtained from GenScript (http://www.genescript.com/, Piscataway, New Jersey, USA).

The bacterial binding assay was done following published protocols [30]. In detail, four types of Gram-negative (E. coli OP50, P. aeruginosa PA14, S. marcescens Db11, S. rubidaea MYb239) and 4 types of Gram-positive bacteria (S. aureus SA113, B. thuringensis Bt247 and MYBt18679, and R. erythropolis MYb53) were grown at 37°C or 28°C overnight in LB to mid-logarithmic phase, pelleted, washed, and resuspended in TBS buffer with CaCl₂ (50 mM Tris, 150 mM NaCl, 2 mM CaCl₂, pH 7.5) at OD₆₀₀ 2. 100 μl bacterial solution was incubated with 6 μg recombinant protein at gentle rotation for 1 h at room temperature. The bacteria were washed three times with 1 ml TBS-CaCl₂ and eluted with 100 μl of 2% SDS. The whole lysates plus 5x loading buffer were heated at 95°C for 5 min, equally loaded onto a 12% SDS-PAGE, analyzed by Coomassie staining, and then transferred to a PVDF transfer membranes (BIO-RAD, Cat. #1704272). After blocking in 5% non-fat milk at room temperature for 1 h the membranes were incubated with Mouse-anti-His mAb (BIO-RAD, Cat. #MCA1396GA) overnight at 4°C, followed by HRP-linked secondary antibody (advansta, Cat. #R-05071-500). The signals were detected using the chemiluminescence phototype-HRP kit (BIO-RAD, Cat. #1705060S) according to the manufacturer’s instructions.