Primary mouse anti his antibody

Purified samples were analysed by SDS-PAGE and western blot. Samples were applied to precast TGX gels (Bio-Rad, Hercules, CA, USA) before staining with InstantBlue Coomassie stain (Sigma-Aldrich) or immediately transferred to PVDF membrane (Bio-Rad) for western blot analysis. Western blots were stained with primary rabbit anti-GNAS (Novus Biologicals, Littleton, CO, USA NBP1-31730), primary mouse anti-His antibody (Qiagen, Hilden, Germany, 34660), secondary goat anti-rabbit 800CW antibody (LI-COR Biosciences, Lincoln, NE, USA, 926–32211), secondary anti-mouse 680CW antibody (LI-COR Biosciences, 926–68070) and an in-house made Alexa Fluor 488 conjugated mouse anti-flag antibody. Western blots were imaged on a Typhoon 5 imaging system (Amersham, Little Chalfont, UK).

The expressed protein was analyzed by SDS-PAGE electrophoresis and Western blotting. For Western blotting analysis, the target gel was transferred onto a 0.45-μm nitrocellulose (NC) membrane (Pall), then the membrane was incubated with primary mouse anti-His antibody (Qiagen) and HRP-conjugated secondary antibody of goat anti-mouse IgG subsequently. Finally, the membrane was visualized by DAB. The quantity of target protein was calculated by Quantity One (Bio-Rad). Cecropin B samples were boiled for 3 min in 2× Tricine sample buffer (Bio-Rad; 161-0739), loaded in 16.5% polyacrylamide Tris/Tricine precast gel, and run at 200 mA for 4.5 h. For Coomassie staining, gels were fixed for 1 h in 12% trichloroacetic acid and 1 h in 40% ethanol and 10% acetic acid, followed by 14 h staining in QC Colloidal Coomassie, and 2 h de-staining in water.