Immunoblotting of Nanobody-Tag Complexes

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting were performed as described previously [34 (link)]. The SDS-PAGE-separated antigens in the gels (4% stacking gel and 12% separating gel) were transblotted onto nitrocellulose membranes (Cytiva, Marlborough, MA, USA), and empty sites on the membranes were blocked with 5% (w/v) skimmed milk (PanReac AppliChem) in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) at room temperature for 1 h. After blocking, the primary antibody (rabbit anti-E-tag or rabbit anti-His tag (Abcam; 1:3000 in TBS-T)) was added to the membranes for 1 h, and then the membranes were washed with TBS-T and incubated with AP-conjugated goat anti-rabbit IgG (Southern Biotech) (1:3000 in TBS-T) for 1 h. BCIP/NBT substrate (SeraCare) was used to detect reactive bands of nanobody–anti-tag complexes.