Bcip nbt substrate

Immunoblotting was used in an attempt to determine reactivity (binding) of the HuscFvs/R9-HuscFvs to the enterovirus produced-VP4. Three hundred microliters of 10⁷ CCID₅₀/0.1 mL of EV71-B5, EV71-C4 and CVA16 were mixed individually with 60 μL of 6 × SDS Protein Loading Buffer and subjected to SDS-PAGE. The separated components were transblotted onto a nitrocellulose (NC) membrane; the blotted NC strips were blocked with 5% skimmed milk and probed separately with 1 μg of HuscFvs/R9-HuscFvs or control (irrelevant) scFv in Tris-buffered saline containing 0.1% Tween-20 (TBST) or buffer alone. Mouse anti-VP4 PAb (1:1,000 dilution) and anti-VP2 MAb (Chemicon; Merck, Millipore, Burlington, MA, United States) (1:3,000 dilution) were used as positive antibody controls. Following incubation and washing, the membranes were incubated with rabbit anti-E-tag antibody (detected HuscFvs/R9-HuscFvs/control scFv). Alkaline phosphatase (AP)-conjugated anti-isotypic antibody and BCIP/NBT substrate (SeraCare Life Sciences, Milford, MA, United States) were used to visualize the reactive bands.

The antigenic preparation was electrophoresed in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) using 4% stacking gel and 12% separating gel cast in Mini-PROTEAN Cell (Bio-Rad, Hercules, California, USA). The SDS-PAGE-separated components in the separating gels were electroblotted onto nitrocellulose membrane (NC) and the unoccupied sites on the blotted NC were blocked with 5% skim milk in Tris-buffered saline containing 0.01% Tween-20 (TBS-T) for 1 h. Excess blocking reagent was discarded; the NC was washed three times with the TBS-T and placed in a solution of mouse mAb to HuscFv34, mouse anti-His (Bio-Rad), or mouse anti-SUMO (Genscript) at RT for 1 h. The NC was washed again with the TBS-T and incubated with goat-anti-mouse Ig-alkaline phosphatase (AP) conjugate (SouthernBiotech, Birmingham, AL, USA) for 1 h. After washing with TBS-T, BCIP/NBT substrate (KPL, SeraCare, Millford, MA, USA) was used to reveal the antigen-antibody reactive bands. The NC was washed with distilled water and air-dried.

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.