Wet transfer unit

The pooled salivary proteins from HV, BB, BC-I, and BC-II subjects of the retrospective cohort were analyzed by SDS-PAGE and subsequently lectin blotting as previously described (Qin et al., 2013 (link), Zhong et al., 2015 (link)). Briefly, For SDS-PAGE, samples were boiled for 4 min at 100 °C mixed with 5 × loading buffer, and run on a 10% polyacrylamide resolving gel and a 3% stacking gel. Molecular mass standards (Thermo Scientific, Waltham, USA) were run with all gels. Some gels were then stained directly with alkaline silver. For lectin blotting, the proteins in gels were then transferred to a PVDF membrane (Immobilon-P; Millipore Corp., Bedford, MA, U.S.A.) with a wet transfer unit (Hoefer Scientific) for 1.5 h at 32 mA. After transfer, the membranes were washed twice with TTBS (150 mM NaCl, 10 mM Tris-HCl, 0.05% v/v Tween20, pH 7.5) and then blocked for 1 h with Carbo-Free Blocking Solution (Vector, Burlingame, CA) at room temperature. The membranes were then washed again and incubated with Cy5 (GE Healthcare, Buckinghamshire, UK) labeled lectins (2 μg/mL in Carbo-Free Blocking Solution) with gentle shaking overnight at 4 °C in the dark. The membranes were then washed twice each for 10 min with TTBS and scanned by red fluorescence channel (635 nm excitation/650LP emission) with the voltage of 800 PMT using a phosphor imager (Storm 840, Molecular Dynamics).