Lectin Blotting to Detect Terminal α2,6 Sialic Acids

Equal amounts of ^SiaIgE or ^AsIgE were resolved on 3–8% Tris-Acetate protein gels (Life Technologies) in SDS-PAGE under nonreducing conditions. For protein stain, gels were incubated in AcquaStain Protein Gel Stain (Bulldog Bio) for 1 h at room temperature and destained in distilled water. For lectin blotting, the protocol was conducted as described^{17 (link)}. Briefly, after resolved proteins on the gel were transferred to Immobilon-PSQ polyvinylidene difluoride membranes (Millipore Sigma), the membranes were blocked with 0.2% BSA in TBS for 1 hour at room temp, washed in TBS, followed by incubation with biotinylated Sambucus nigra lectin (SNA; 0.4 μg/ml; Vector Laboratories) in TBS with 0.1 M Ca²⁺ and 0.1 M Mg²⁺ for 1 hour at room temp to determine the level of terminal α2,6 sialic acids on N-linked glycans of proteins. The membrane was then washed in TBS and incubated with alkaline phosphatase conjugated goat anti-biotin (1:5000 dilution; Vector Laboratories) in TBS for 1 hour at room temp. Sialylated proteins on membranes were visualized by incubation with 1-Step NBT/BCIP plus Suppressor Substrate Solution.