Rabbit anti his antibody

The binding of protein to carbohydrate component of C. difficile cell wall was performed as previously described (44 (link)). Briefly, 5 μL of 5 mg/mL of PG-PS, PG, and PS samples as well as 5 μM 6×His-CBD and bovine serum albumin (BSA) were spotted onto a nitrocellulose membrane (Bio-Rad) and dried for 30 min. Non-specific binding was blocked in 2% (wt/vol) BSA in Tris buffered saline with Tween 20 (TBST) (25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20) at room temperature for 1 h. The membrane was pre-equilibrated in 50 mM Tris-HCl (pH 7.4) at room temperature for 20 min and subsequently incubated with or without 6×His-CBD at room temperature with agitation for 1 h. Membranes were washed with TBST buffer at room temperature for 30 min before incubated with 1:1,000 rabbit anti-His antibody (Cell signaling) at room temperature overnight. Anti-rabbit IgG conjugated to HRP antibody, 1:500, (Cell signaling) was applied and incubated for 1 h, followed by adding of ECL substrate (Bio-Rad). The signal was detected using gel imaging instrument (Syngene G: Box F3 gel Documentation System; New England BioGroup [G:Box F3]).