Dectin 1 antibody

Immunoblotting for Dectin-1 was performed using an equal protein 30 μg of cell lysate or recombinant protein per lane. Samples were loaded on a 4%–12% precast NuPAGE Bis-Tris polyacrylamide gel (Invitrogen).^{55–58 (link)} Proteins were electrophoresed using MES-SDS buffer at 200 V for 35–45 minutes per the manufacturer's instructions (Invitrogen). Native gel electrophoresis was performed using a Novex 4% Tris-glycine gel (Invitrogen). Samples were mixed with a cold native gel sample buffer (Invitrogen) before loading. Electrophoresis was run at room temperature at a constant voltage of 80 V for 5 hours. Proteins were then transferred from the gel to a nitrocellulose membrane using the XCell II Mini-Cell and sandwich blot module (Invitrogen) in Tricine-10% methanol-0.01% SDS transfer buffer (Invitrogen) at 30 V for 1 hour. Blots were blocked for 1 hour at room temperature with 10% nonfat milk and then incubated with primary Dectin-1 antibody (R&D system) (1:500 in 1% nonfat milk) overnight at 4°C. Blots were then incubated with 1:5000 goat anti-rabbit IgG-horseradish peroxidase for 2 hours at room temperature. Signal was detected using the enhanced chemiluminescence kit (Amersham), and blots were exposed to Kodak Biomax MS film.