2.4 Western blotting
Baculovirus-infected Sf21 supernatants were mixed with 6X non-reducing
buffer (0.1 M Tris-HCl, pH 6.8, 0.2% bromophenol blue, and 20%
glycerol). Protein samples were then loaded onto 4-15% Mini-PROTEANĀ®
TGX Stain-Free gels (Bio-Rad) and separated at 200V for 30 mins.
Proteins were transferred onto PVDF membranes (Bio-Rad) using the
Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were probed with
mouse anti-His (clone 27471001, GE Healthcare, Chicago, IL, US) followed
by goat anti-mouse IgG HRP (clone NA9310N; Cell Signaling Technology,
Danvers, MA, US). PVDF membranes were developed using Amersham ECL
Western Blotting Detection Reagent (GE Healthcare) and chemiluminescence
was detected using a ChemiDoc Imager (Bio-Rad).