Wet electroblotting apparatus

The affinity of the AviPure for IgG was investigated using protein affinity binding. Samples collected during cultivation (before and after induction) were resolved by a 15 % SDS-PAGE gel. Separated proteins were blotted onto nitrocellulose membranes (Whatman, Dassel, Germany) using a wet electro-blotting apparatus (Bio-Rad, USA). To prevent non-specific binding, the free binding sites of the membranes were saturated with 5 % fat free dry milk powder in TTBS (overnight at 4 °C with gentle agitation). Thereafter, the membrane was thoroughly washed five times, each for 5 min with 50 mL TTBS. After each incubation, the membrane was washed in the same way. The Goat anti-protein A polyclonal antibodies HRP conjugate at dilution of 1: 6000 was applied to the membrane, incubated for 1.5 h at room temperature with gentle agitation. This was followed by washing and the membranes were washed again and the proteins were subsequently visualized with a chemiluminescence kit (ECL) on X-ray film.

SDS-PAGE analysis was performed using 12 % gels according to the standard method. The entire gel was stained in Coomassie blue staining solution overnight before being placed in destaining buffer. For Western blotting, the proteins on the gel were transferred to a polyvinylidene difluoride membrane (Immobilon P, Millipore) using a wet electroblotting apparatus (Bio-Rad) at 100 V for 50 min in a solution of Tris-glycine (25 mM Tris, 192 mM glycine). The membrane was blocked by incubating with 5 % non-fat milk for 1 h at RT. Then, the membrane was immunoblotted with primary antibody at 4 °C overnight and with HRP-conjugated secondary antibodies for 1 h at RT. Detection of the bound antibody was performed using Super Signal West Pico Chemiluminescent Substrate (Pierce). The primary antibody against CD40-N (AF632) was purchased from R&D Systems. The anti-Flag antibody was purchased from Sigma (F3165). Mouse monoclonal antibodies against CD40 (G28-5 and 3A8) were purchased from ATCC. Anti–NF-κB2 (#4882) was purchased from Cell Signaling Technology (Danvers, MA). Anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody was purchased from Kangchen (KC-5G4, Shanghai, China).